Nyvac-based plasmodium malaria vaccine

ABSTRACT

The disclosure generally provides a WRrNYVAC comprising multiple antigens from different stages of a malaria parasite ( Plasmodium ) life cycle, methods for producing them, immunogenic vaccine formulations comprising WRrNYVAC, such as  P. vivax  containing formulations (WRPvrNYVAC) and  P. falciparum  containing formulations (WRPfrNYVAC) and methods for the prevention and/or treatment of malaria infections and poxvirus infections.

CORRESPONDENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Applications 62/523,708 and 62/638,442 filed respectively on Jun. 22, 2017 and Mar. 5, 2018 wherein the contents of the applications and related appendices are incorporated herein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS OR INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support from the Walter Reed Army Institute of Research, a subordinate organization of the United States Army Medical Research and Materiel Command. The United States government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

A sequence listing is included herein.

BACKGROUND

Malaria remains one of the world's most prevalent serious infectious diseases. In 2015, 95 countries and territories retained ongoing malaria transmission and an estimated 3.2 billion people—nearly half the world's population—were at risk of malaria (WHO, 2016). Approximately 214 million cases worldwide in 2015 and an estimated 438,000 million deaths per year (WHO, 2016) existed. Mortality is primarily in children under the age of five and in pregnant women. Every 45 seconds, an African child dies of malaria. The disease is transmitted from person to person by infected mosquitoes. Past eradication efforts involved massive insecticide campaigns. These proved successful in the Southeast United States. But, these efforts failed in most poorly developed tropical countries. Current efforts involve distribution of mosquito nets, particularly mosquito nets impregnated with insecticide, to prevent mosquito bites at night. During the past 15 years, coverage of mosquito control interventions increased substantially in Africa. In 2014, more than half of the population at risk in Africa (56%) had access to an insecticide-treated mosquito net, compared to 2% in 2000 (WHO, 2016). However, emerging parasite resistance to antimalarial medicines and mosquito resistance to insecticides, if left unaddressed, could render some of the current tools ineffective and trigger a rise in global malaria mortality. If an individual is treated, convalescent time can be between 5-20 days. Specifically for Plasmodium vivax (Pv) malaria, infections can reoccur months to years later from the original infection based on the unique biology of Pv malaria parasites forming hypnozoites that remain dormant in liver cells. Currently, there is no effective, durable malaria vaccine available for Pv malaria. The need for a malaria vaccine remains critical for protection of millions of people from this disease.

Malaria caused by Pv remains a major public health threat, especially in Africa, Thailand, South America, South East China, and the Koreas among children and pregnant women. Pv is the most frequent and widely distributed cause of recurring (Benign tertian) malaria, it is one of the five species of malaria parasites that commonly infect humans. Although it is less virulent than Plasmodium falciparum, the deadliest of the five human malaria parasites, Pv malaria infections can lead to severe disease and death, often due to splenomegaly (a pathologically enlarged spleen). Pv is carried by the female Anopheles mosquito, since it is only the female of the species that bites. An effective malaria vaccine offers a valuable tool that reduces the disease burden. It could also contribute to elimination of malaria in some regions of the world. Current malaria vaccine candidates are directed against human and mosquito stages of the parasite life cycle, but thus far, relatively few proteins have been studied for potential vaccine development.

SUMMARY

To date there have been no therapeutically effective compositions for administering to subjects that provide a protective response to Plasmodium sp. infections. While others have created rNYVAC systems expressing more than one Plasmodium gene, the rNYVAC compositions as administered to subjects did not produce a therapeutic and protective effect to the subject receiving the composition. Therefore, therapeutically effective and protective vaccine remain to be identified. Provided here is are recombinant NYVAC containing compositions capable of expressing Plasmodium sp. genes across all stages of the Plasmodium life cycle and a method of administering the composition that that provides a therapeutic and protective benefit to the subject. Also provided is a novel promoter for use in expressing the Plasmodium sp. genes of interest.

Therefore provided here is a method of eliciting a protective immune response in a subject against a malaria infection, comprising administering to the subject an effective amount of a recombinant NYVAC (rNYVAC) virus capable of expressing a malaria antigen gene, wherein the malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: A1 and is inserted into a region (locus) of the NYVAC viral genome selected from the group consisting of A26L, A56R, 14L, J2R, B13/B14R, and C7L-K1L, and wherein the malaria antigen gene encodes a malaria antigen selected from the group consisting of a pre-erythrocytic stage antigen, a blood stage antigen, or a transmission blocking stage antigen.

The rNYVAC virus of the method when administered to a subject can elicit a protective immune response against a poxvirus infection in the subject. The rNYVAC virus is capable of expressing two or more malaria antigen genes encoding malaria antigens of different developmental stages. The number of malaria genes expressed by the rNYVAC virus include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes from one or more Plasmodium sp.

Another methods contemplates administering two or more (any number between 2 to 30) recombinant NYVAC viruses are administrated, wherein the two or more rNYVAC viruses are capable of expressing two or more malaria antigen genes encoding malaria antigens of different developmental stages.

The method of administration for the rNYVAC virus compositions described include either in combination or separately, via a route selected from the group consisting of (skin) scarification, intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, oral administration, and intranasal administration. Preferably, the route of administration is skin scarification followed by intramuscular injection of the formulation.

The method of any of the above contemplates administering the rNYVAC virus to the subject as a vaccine formulation. The isolated purified rNYVAC virus can be lyophilized and stored in glass vials at room temperature and even at higher tropical temperatures and remain stable.

The contemplated methods for the rNYVAC viruses described and their use include rNYVAC wherein the malaria antigen gene is selected from the group consisting of AMA1, CelTOS, CS, LSA1-RPTLS, SIAPL, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45 of Plasmodium falciparum. A P. falciparum antigen gene can be inserted into a region of the rNYVAC viral genome selected from the group consisting of A26L, A56R, I4L, J2R, and B13/B14R regions. When a WRPfrNYVAC construct containing one up to 25 of these genes is administered to a subject, the construct elicits a protective immune response against a P. falciparum infection in said subject.

Another method and construct contemplated includes administering to the subject an effective amount of five to thirty rNYVAC viruses, wherein the five to thirty rNYVAC viruses jointly express P. falciparum antigen genes of AMA1, CelTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45. The method can be wherein altogether 25 rNYVAC viruses are administered to the subject, wherein each of the 25 rNYVAC viruses express at least one different P. falciparum malaria antigen gene.

Another method and rNYVAC contemplated comprises administering to the subject an effective amount of two to seven or 15 rNYVAC viruses, wherein the two to seven or 15 rNYVAC viruses jointly express at least P. falciparum antigens AMA1, CelTOS, LSA1-RPTLS, TRAP, and Pfs25.

Also contemplated are methods and rNYVAC viruses described herein, wherein the malaria (Plasmodium) antigen gene is selected from the group consisting of: CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25 of Plasmodium vivax, and wherein administering the rNYVAC viruses elicits a protective immune response against a P. vivax infection in the subject.

Another method and composition contemplates administering to the subject an effective amount of one to eight rNYVAC viruses, wherein the one to eight rNYVAC viruses jointly express P. vivax antigens of CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25.

The method contemplates an rNYVAC, wherein altogether eight rNYVAC viruses are administered, wherein each of the one to eight recombinant NYVAC viruses individually expresses at least one different P. vivax malaria antigen.

Another method contemplated includes a recombinant NYVAC virus is constructed by the steps of:

-   -   a) preparing a shuttle plasmid comprising an expression cassette         flanked by about 500 bp sequences upstream and downstream of the         NYVAC A26L, A56R, 14L, J2R, B13/B14R, or C7L-K1L gene, wherein         the expression cassette comprises the malaria antigen gene under         the control of the compact synthetic early-late promoter;     -   b) transfecting the shuttle plasmid to host mammalian cells and         co-infecting the host mammalian cells with a parent NYVAC virus         to allow in vivo recombination; and     -   c) screening for a rNYVAC virus obtained from step b) to obtain         the recombinant NYVAC virus, wherein the expression cassette is         located in the rNYVAC genome.         The shuttle plasmid can further comprise an E. coli gpt gene.         This method can further comprise using a recombinant NYVAC virus         as the parent NYVAC virus to obtain a second recombinant NYVAC         virus capable of expressing two or more malaria antigen genes.

A recombinant NYVAC virus is contemplated that is capable of expressing a Plasmodium falciparum malaria antigen gene selected from the group consisting of: AMA1, CelTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45, wherein the P. falciparum malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: 1 and is inserted into a region of the NYVAC viral genome selected from the group consisting of: A26L, A56R, 14L, J2R, and B13/B14R. The rNYVAC virus can be capable of expressing up to five P. falciparum malaria antigen genes, each of which is inserted into a different region of the NYVAC viral genome.

A vaccine formulation is included that comprises a rNYVAC virus described above, wherein the vaccine formulation is capable of eliciting a protective immune response against a P. falciparum infection when administered to a subject. The vaccine formulation can comprise 8 to 25 or 8 to 30 recombinant NYVAC viruses, each of which expresses at least one different P. falciparum malaria antigen gene. Alternatively, the vaccine formulation can comprise two to seven recombinant NYVAC viruses, wherein the two to seven recombinant NYVAC viruses jointly express at least P. falciparum antigens AMA1, CelTOS, LSA1-RPTLS, TRAP, and Pfs25.

Also disclosed is a recombinant NYVAC virus capable of expressing a Plasmodium vivax malaria antigen gene selected from the group consisting of: CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25, wherein the P. vivax malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: 1 and is inserted into a region of the NYVAC viral genome selected from the group consisting of A26L, A56R, I4L, J2R, B13/B14R, and C7L-K1L. The rNYVAC virus can be capable of expressing eight P. vivax malaria antigen genes, wherein three P. vivax malaria antigen genes are inserted in C7L-K1L region of the NYVAC viral genome.

A vaccine formulation is contemplated comprising the recombinant NYVAC virus of either claim 23 or claim 24, wherein the vaccine formulation is capable of eliciting a protective immune response against a P. vivax infection when administered to the subject. The vaccine formulation can comprise eight recombinant NYVAC viruses, each of which expresses at least one different P. vivax malaria antigen gene.

For any of the methods of administering the formulation the subject can be human, however the subject can be any animal infected with Plasmodium sp.

Also contemplated are the following materials and methods of making and using recombinant NYVAC viruses expressing P. falciparum antigens that can also be used to protect a subject from a pox virus. The following materials and methods can be also used with other Plasmodium sp. genes.

1. A method of making a recombinant NYVAC viral vector (“WRrNYVAC”) comprising the steps of:

(i) generating an expression cassette comprising a genomic or a cDNA copy of one or more genes encoding 25 multistage P. falciparum antigens wherein the gene is under control of a poxvirus promoter;

(ii) subcloning the expression cassette into a NYVAC donor plasmid to create a shuttle plasmid wherein the shuttle plasmid is inserted into A26L, A56R, I4L, J2R, or B13/B14R sites in the NYVAC donor plasmid genome;

(iii) transfecting and simultaneously co-infecting with parental NYVAC to promote in vivo recombination;

wherein WRrNYVAC is generated to elicit immunity directed against multiple stages in a malarial life cycle.

2. A recombinant NYVAC viral vector (WRrNYVAC) according to the method of claim 1 comprising:

(i) A NYVAC donor plasmid;

(ii) a left recombination arm comprising approximately 500 base pairs upstream of an open reading frame of the NYVAC donor plasmid genome;

(iii) a right recombination arm comprising approximately 500 base pairs downstream of the ORF of the NYVAC donor plasmid genome;

(iv) a gene inserted between the left recombination arm and the right recombination arm that is selected from the group consisting of 25 multistage P. falciparum genes: AMA1, celTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45;

(v) a poxvirus promoter;

(vi) an E. coli gpt gene encoding xanthine guanine phosphoribosyl transferase; and

(vii) a B-lactamase gene encoding for ampicillin resistance;

-   -   wherein the recombinant NYVAC vector expressing 25 P. falciparum         antigens elicits immunity directed against multiple stages in a         malarial life cycle.

3. The WRrNYVAC vector of claim 2, wherein the poxvirus promoter is a compact synthetic early-late promoter.

4. A vaccine formulation comprising the vector of claim 3 for use in the prevention or treatment of malaria, comprising a plurality of malaria-derived antigens.

5. The vaccine formulation according to claim 4, wherein the P. falciparum malaria antigens selected from the pre-erythrocytic stage include: AMA1, celTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, and TRAP.

6. The vaccine formulation of claim 3, wherein the P. falciparum malaria antigens selected from the blood stage include: EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, and Rh5.

7. The vaccine formulation of claim 3, wherein the P. falciparum malaria antigens selected from the transmission blocking stage include: Pfs16, Pfs25, Pfs28, and Pfs48.45.

8. A method for immunizing a subject against malaria or poxvirus, wherein the method comprises administering to the subject an effective amount of the vaccine formulation of claim 4.

9. A method of eliciting a protective immune response in a subject against malaria or poxvirus infection comprising administering the vaccine formulation of claim 4 to a human.

10. The method of any of claims 8 or 9, wherein the administering is via a route of scarification followed by a boost via intramuscular injection. While the method can utilize other methods of administration such as subcutaneous, intradermal, intramuscular alone or in combination with skin scarification, for optimal immune response the method is skin scarification followed by intrasmuscluar injection.

11. A method of treating malaria or poxvirus in a subject, comprising administering an effective amount of the vector according to claim 4 to the mammal in need thereof.

12. A method of treating malaria or poxvirus in a subject, comprising administering a priming vaccine formulation comprising at least one first malaria antigen; and a boosting vaccine formulation comprising at least one second malaria antigen, wherein at least one of the formulations comprises the vector according to claim 4 to the mammal in need thereof.

13. A kit comprising a vaccine formulation comprising the vector according to claim 4.

Also contemplated are the following materials and methods of making and using recombinant NYVAC viruses expressing P. vivax antigens that can also be used to protect a subject from a pox virus. The materials and methods described for producing a P. vivax expressing NYVAC can be utilized with other Plasmodium sp.

1. A method of making a recombinant NYVAC viral vector (“WRPvrNYVAC”) comprising the steps of:

(iv) generating an expression cassette comprising a synthetic DNA copy of one or more genes encoding 8 multistage P. vivax antigens wherein the gene is under control of a poxvirus promoter;

(v) subcloning the expression cassette into a NYVAC donor plasmid to create a shuttle plasmid wherein the shuttle plasmid is inserted into A26L, A56R, I4L, J2R, B13/B14R sites, or C7L-K1L region in the NYVAC donor plasmid genome;

(vi) transfecting and simultaneously co-infecting with parental NYVAC to promote in vivo recombination;

wherein WRPvrNYVAC is generated to elicit immunity directed against multiple stages in a malarial life cycle.

2. A recombinant NYVAC viral vector (WRPvrNYVAC) according to the method of claim 1 comprising:

(viii) A NYVAC donor plasmid;

(ix) a left recombination arm comprising approximately 500 base pairs upstream of an open reading frame of the NYVAC donor plasmid genome;

(x) a right recombination arm comprising approximately 500 base pairs downstream of the ORF of the NYVAC donor plasmid genome;

(xi) a gene inserted between the left recombination arm and the right recombination arm that is selected from the group consisting of 8 multistage P. vivax genes: CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25;

(xii) a poxvirus promoter;

(xiii) an E. coli gpt gene encoding xanthine guanine phosphoribosyl transferase; and

(xiv) a B-lactamase gene encoding for ampicillin resistance;

wherein the recombinant NYVAC vector expressing 8 P. vivax antigens elicits immunity directed against multiple stages in a malarial life cycle.

3. The WRPvrNYVAC vector of claim 2, wherein the poxvirus promoter is a compact synthetic early-late promoter.

4. A vaccine formulation comprising the vector of claim 3 for use in the prevention or treatment of malaria, comprising a plurality of malaria-derived antigens.

5. The vaccine formulation according to claim 4, wherein the P. vivax malaria antigens selected from the pre-erythrocytic stage include: CS-VK210, CS-VK247, and TRAP-SSP2.

6. The vaccine formulation of claim 3, wherein the P. vivax malaria antigens selected from the blood stage include: AMA1, MSP1 fragment p42, and Duffy Binding Protein region II (DBP RII).

7. The vaccine formulation of claim 3, wherein the P. vivax malaria antigens selected from the transmission blocking stage include Pvs 28 and Pvs 25.

8. A method for immunizing a subject against malaria or poxvirus, wherein the method comprises administering to the subject an effective amount of the vaccine formulation of claim 4.

9. A method of eliciting a protective immune response in a subject against malaria or poxvirus infection comprising administering the vaccine formulation of claim 4 to a human.

10. The method of any of claims 8 or 9, wherein the administering is via skin scarification followed by an intramuscular boost vaccine formulation. While the method can utilize other methods of administration such as subcutaneous, intradermal, intramuscular alone or in combination with skin scarification, for optimal immune response the method is skin scarification followed by intrasmuscluar injection.

11. A method of treating malaria or poxvirus in a subject, comprising administering an effective amount of the vector according to claim 4 to the mammal in need thereof.

12. A method of treating malaria or poxvirus in a subject, comprising administering a priming vaccine formulation comprising at least one first malaria antigen; and a boosting vaccine formulation comprising at least one second malaria antigen, wherein at least one of the formulations comprises the vector according to claim 4 to the mammal in need thereof.

13. A kit comprising a vaccine formulation comprising the vector according to claim 4.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 depicts Western Blot gels illustrating protein expression from WRrNYVAC β-actin as a loading control. The figure displays P. falciparum expression of various infected cells (Mock, wtNYVAC, WRr-NYVAC and NYVAC-Pf7 (as described in Tine et al.)). Expression of all the proteins was observed for the WRrNYVAC, but as expected not in the mock and wtNYVAC lanes. Expression in the NYVAC-Pf-7 system only was observed for AMA-1, CSP, and TRAP proteins, which are all pre-erythrocytic proteins. Recombinant viruses, WRrNYVAC, expressing these proteins have also been developed and have been shown to express in BHK-21 cells (LSA-RPTLS, MSP1-p42, MSP3, MSP5, MSP7, MSP9, Pfs25 and Pfs48.45) (data not shown).

FIG. 2A-2E is a bar graph illustrating assessment of viral replication where confluent baby hamster kidney cells (BHK-21) (FIG. 2A); (FIG. 2B) human keratinocyte (hK); (FIG. 2C) human dermal fibroblasts (hDF); (FIG. 2D) human dermal microvascular endothelial cells (hMVEC-D); and (FIG. 2E) human skeletal muscle cells (hSM) monolayers were Mock infected, infected with wild-type NYVAC (wtNYVAC), WRrNYVAC expressing a Pf transgene, or NYVAC-Pf7 at a low multiplicity of infection (MOI). Multi-cycle growth curves of NYVAC illustrate where the indicated cell lines were infected at an MOI of 0.05 with wtNYVAC, NYVAC-Pf7 or WRrNYVAC expressing CSP in an effort to determine if NYVAC is replication competent or replication abortive in each cell line.

FIG. 3A-3E depicts Western Blot gels illustrating protein expression corresponding to multi-cycle growth curves of NYVAC where the indicated cell lines were infected at an MOI of 0.05 with wtNYVAC, NYVAC-Pf7, or WRrNYVAC expressing CSP over the course of 4 days. FIG. 3A to E respectively depicts BHK cells, hK cells, hDF cells, hDMEC cells, and hSK cells.

FIG. 4A-4B are bar graphs illustrating antibody response to PfCSP with a (a) single immunization and (b) prime-boost.

FIG. 5 is a bar graph illustrating TNF-α response. Single immunization using either skin scarification (“SS”) or intramuscular (“IM”) appears in FIG. 4A which shows the endpoint titer of TNF-α of 5 mice immunized by SS only or by IM only. FIG. 4B depicts the amount of TNF-α (reciprocal endpoint titer) of the mouse was detected using a Luminex 200 instrument and a cytokine 10-Plex mouse panel kit (Invitrogen; Cat. No. LMC0001M per manufacturer's instructions) after the initial prime administration but before the boost injection. TNF—The sequence of prime boost administration is indicated with either SS or IM being first. The pre-boost bleed was taken at Day 25 after administration of the prime. The terminal bleed of the mouse was taken at Day 50 after administration of the prime. There was no detectable TNF-α for SS→SS (skin scarification of both the prime and boost). The greatest amount of TNF-α was detected for the terminal bleed performed by IM→IM (IM injection for both prime and boost).

FIG. 6 is a bar graph illustrating IFN-γ response measured in pg using a cytokine 10-Plex mouse panel kit (Invitrogen). There were sham animal controls that were injected with mock infected BHK-21 cells. Then animals were treated with WRrNYVAC with either single administrations by skin scarification (SS) or intramuscularly (IM) or by prime and boost administration of: SS→SS, IM→IM, SS→IM, IM→SS, or IM→IM.

FIG. 7A-7B. Protein translation of WRPvrNYVAC expressing P. vivax (Pv) proteins in baby hamster kidney-21 (BHK-21) and primary human dermal fibroblasts cells (hDF). Confluent 35 mm dishes of either BHK-21 or hDF cells were infected at an MOI of 5.0 with either mock, wild-type NYVAC (wtNYVAC), recombinant NYVAC expressing P. vivax (WRPvrNYVAC), or CS-VK247 (CSP gene variants VK210 or VK247). Cells were harvested over 24 hours post infection. Western blots were performed with specific anti-PvCS-210 or anti-PvCS-247 antibodies. As expected, no protein was detected in the mock (negative control) as well as in the wtNYVAC control. Substantial amounts of CSP protein were detected in WRPvrNYVAC expressing cells using either antibody. Recombinant viruses, WRrNYVAC, have been developed expressing P. vivax TRAP-SSP2, AMA-1, MSP1-p42, DBT, RII, Pvs25, and Pvs28 and have been demonstrated to be expressed in BHK-21 cells (data not shown).

FIG. 8A-8B. Multi-cycle replication and protein translation of WRPvrNYVAC expressing Pv proteins in primary human dermal fibroblasts cells. Briefly, five sets of confluent 35 mm dishes were infected in duplicate at an MOI of 0.05 of either mock, wild-type NYVAC (wtNYVAC), or recombinant NYVAC expressing Pv (WRPvrNYVAC) CS gene variants VK210 or VK247. Cells were harvested over 4 days (Days 1, 2, 3 and 4) according to the assay procedure. FIG. 8A. Plaque assays were performed to determine the replication rate of the virus over 4 days. FIG. 8B. Western blots were performed to determine protein translation over 4 days using the same antibodies against CSP as discussed for FIG. 7.

FIG. 9. The process of producing the pMPΔVACV locus::Pf or Pv gene that is ready for NYVAC in vivo recombination starting with pMPAE3L::MCS (which is what from where????). To obtain pMP, the pMPΔVACV is digested with SacIIl and XmaI restriction enzyme to remove the E3L recombination arms and then the fragments are treated with phosphatase (STEP 1). STEP 2 requires one to PCR the sequences 500 bp upstream and 500 bp downstream of the following gene loci: (1) A56R, (2) J2R, (3) A26L, (4) I4L, (5) B13/14R and (6) C7L-K1L. “LRA” stands for left recombination arm and “RRA” stands for right recombination arm. STEP 3 requires a restriction enzyme digest of all three fragments with either SacII or PluTI and XmaI. Then ligate digested (1) A56R, (2) J2R, (3) A26L, (4) I4L, (5) B13/14R and (6) C7L-K1L. LRA through RRA PCR fragment into digested pMP plasmid. In STEP 4, whole plasmid PCR is performed to remove (1) A56R, (2) J2R, (3) A26L, (4) I4L, (5) B13/14R and (6) C7L-K1L genes between the LRA and RRA leaving the recombination arms intact to produce pMP::VACV locus. For STEP 5, once the PCR is complete, the complex is digested with DPNI (a NRB restriction enzyme) overnight to remove the template plasmid. The template plasmid is used to transform competent bacterial cells, such as but not limited to E. coli. Bacterial colonies are isolated to prepare mini-preps according to regular procedures, such as those taught in Sambrook's Molecular Cloning. Diagnostic restriction enzyme digests are performed to identify the new clones and the plasmids are sent out for sequencing for sequence confirmation of pMPΔVACV locus::MCS. For STEP 6, the pMPΔVACV locus::MCS is digested and treated with phosphatase. STEP 7 requires that the P. falciparum (Pf) 3D7 genes from Pf genomic DNA is PCR-ed to reverse transcribe the mRNA. For P. vivax (Pv), the genes were synthetically derived under direction by GENEWIZ. The Pf and Pv genes of interest are digested with restriction enzymes. The Pf or Pv genes are ligated into the plasmid per standard procedures to produce the plasmid containing the Pf or Pv genes, pMPΔVACV locus::Pf or Pv genes. The plasmid can now be used to transform competent bacterial cells. Colonies can be isolated from the transformed bacterial cells, and diagnostic restriction enzyme digests are performed to identify complete plasmids. The “complete plasmids” are sent out for sequencing to confirm. The complete confirmed plasmids are then ready for NYVAC in vivo recombination.

FIG. 10. This depicts a model recombinant NYVAC (rNYVAC) genome to be used with P. falciparum genes. The Pf genes indicated under 14L, J2R A26L, Af6R and B13R/B14R loci can be inserted in those sites alone or in an operably linked combination with additional Pf genes as long as the inserted genes are not greater than 3,000 bp in size. The genes can be placed in any order relative to each other and can be a gene expressed in any of the 3 stages of the Plasmodium life cycle. For the C7L-K1L loci, again a Pf gene can be inserted up at this site alone or in operable linkage with other Pf genes up to 3,000 bp. Alternatively, an exemplary shuttle plasmid that can be used is depicted in FIG. 11. Such a shuttle plasmid can be inserted in the C7L-K1L locus (or the other NYVAC loci of 14L, J2R, A26L, A56R, and B13/B14R), wherein the shuttle plasmid contains one or more genes Pf genes and wherein the combination of Pf genes cannot exceed 9,000 bp in the shuttle plasmid. The shuttle plasmid can introduce more genes into a locus, such as C7L-K1L, than if the genes were inserted alone.

FIG. 11. The figure is a depiction of the shuttle plasmid, pMPΔCTL-K1Lx3MCS, and its components. “PLRA” represents the plasmid left recombination arm. “PRRA” represents the plasmid right recombination arm. “MCS1” represents multiple cloning site 1. “MCS2” represents multiple cloning site 2. “MCS3” represents multiple cloning site 3. “IGR1” represents vaccinia virus intergenic site 1. “IGR2” represents vaccinia virus intergenic site 2. The shuttle plasmid is designed to insert up to 3 genes (each up to a size of 3 kilobases, kpb) within the 3 unique multiple cloning sites in the plasmid; therefore, the shuttle plasmid can contain up to 9,000 bp of Pv or Pf genes in any order and from any stage. The genes are separated by intergenic regions, which contain two vaccinia virus transcription termination sites ([T]₅NT). For P. vivax, the WRPvNYVAC can simultaneously express 8 P. vivax proteins from 6 NYVAC loci and can utilize the pMPΔCTL-K1Lx3MCS plasmid in the CTL-K1L loci of the rNYVAX genome. The P. vivax Duffy Binding Protein Region II (DBP, RII) in MCS1, Pvs25 in MCS2, and Pvs3 in MCS3. For P. falciparum, the rNYVAC specific for P. falciparum can also utilize the shuttle plasmid, and containing up to 3 genes (each gene or gene combination up to a size of 3 kbp).

FIG. 12. The figure depicts an exemplary WRrNYVAC constructed to express P. vivax genes. A similar construct can be prepared using other Plasmodium sp. genes. The P. vivax gene for AMA1 (1760 bp) is inserted at the I4L locus. The P. vivax CS-VK210 gene (1063 bp) is inserted at the J2R locus. The P. vivax TRAP/SSP2 gene (1742 bp) is inserted at the A26L locus. The P. vivax MSP1-p42 gene (2168 bp) is inserted at the B13R/B14R locus. Three genes using a shuttle plasmid are inserted at the C7L-K1L locus (DBP, RII, Pvs25 and Pvs28). This WRPvrNYVAC stably expresses eight P. vivax genes in one genome.

FIG. 13. Pre-erythrycytic growth genes are assessed in dermal fibroblasts and skeletal muscle cells. Cells are cultured at subconfluency in 12-well plates as described. They are infected in triplicate at an MOI of 0.05 (e.g., a low MOI, or 1 virus per 20 cells) of the indicated virus. Well 1 is harvested at 2 hours post infection. Subsequent harvests are performed at 24, 48 and 72 hours post infection. Harvest the virus and the virus is tittered on BSC40 cells. The number of plaques are counted to calculate the percent change in replication from the input virus. Data is shown as an average of the experiment performed in triplicate.

FIG. 14. Blood stage growth genes are assessed in dermal fibroblasts and skeletal muscle cells. Cells are cultured at subconfluency as described in 12-well plates. They are infected in triplicate at an MOI of 0.05 (e.g., a low MOI, or 1 virus per 20 cells) of the indicated virus. Well 1 is harvested at 2 hours post infection. Subsequent harvests are performed at 24, 48 and 72 hours post infection. Harvest the virus and the virus is tittered on BSC40 cells. The number of plaques are counted to calculate the percent change in replication from the input virus. Data is shown as an average of the experiment performed in triplicate.

FIG. 15. Transmission-blocking growth is assessed in dermal fibroblasts and skeletal muscle cells. Cells are cultured at subconfluency as described in 12-well plates. They are infected in triplicate at an MOI of 0.05 (e.g., a low MOI, or 1 virus per 20 cells) of the indicated virus. Well 1 is harvested at 2 hours post infection. Subsequent harvests are performed at 24, 48 and 72 hours post infection. Harvest the virus and the virus is tittered on BSC40 cells. The number of plaques are counted to calculate the percent change in replication from the input virus. Data is shown as an average of the experiment performed in triplicate.

FIG. 16. FIG. 16A depicts an exemplary pMPΔA26L::MCS, wherein the A26L locus is deleted and into which is a multiple cloning site (MCS). FIG. 16B depicts an pMPΔA56R::MCS. FIG. 16C depicts an pMPΔB13/BI4R::MCS. FIG. 16D depicts an pMPΔI4L::MCS. FIG. 16E depicts pMPΔJ2R::MCS. FIG. 16F depicts pMPΔC7L-K1L::MCS. Into the MCS domains of these plasmids can be placed a Plasmodium gene of interest. Once the plasmids depicted in FIG. 16 contain a Plasmodium gene of interest in the MCS site, they can then be used as shown in FIG. 9.

DETAILED DESCRIPTION

Malaria is the condition induced by a parasitic infection of a human or other animal (e.g. mouse). Examples of the parasitic organisms that cause malaria include, but are not limited to, Plasmodium (P.) species including P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii.

It is desirable to formulate a vaccine formulation that comprises multiple antigens from different stages of the parasite life cycle. In doing so, the inability to mount a fully effective immune response to a particular antigenic component of the vaccine formulation or to antigens of a given stage of the life cycle may be compensated by effective responses to other antigens or life cycle stages, resulting in protective immunity. In the late 1990's, a PhaseI/IIa human malaria challenge clinical trial was conducted to test a recombinant NYVAC expressing seven multi-antigen, multi-stage genes from P. falciparum (NYVAC-Pf-7) as a malaria vaccine candidate (Ockenhouse et al., 1998). Volunteers were given a series of three intramuscular injections with NYVAC-Pf-7 and challenged with P. falciparum through Anaopholes stephensi mosquito bites. Thirty-four out of 35 vaccinated individuals demonstrated delay to parasitemia while the remaining volunteer demonstrated sterile protection compared to the control volunteers (Ockenhouse et al., 1998).

The NYVAC-Pf-7 results were optimistic because protection and delay to parasitemia of volunteers was attained with a recombinant NYVAC construct having limited promoter activity with less than ideal assembly indicative of ineffective transcription and resulting in decreased translation of P. falciparum proteins. NYVAC is a vaccinia virus that is a derivative of the Copenhagen strain of vaccinia virus. NYVAC has had 18 open reading frames (ORFs) deleted that contributes to its attenuation. Furthermore, the NYVAC intramuscular route of immunization was not optimal, because vaccinia virus (VACV) has historically been administered by percutaneous administration (i.e., scarification) to protect against smallpox with immunity lasting for decades leading to the eventual eradication of smallpox disease (Liu et al. 2010; McClain et al. 1997; Stewart et al. 2006).

The global proteomic evaluation of P. falciparum from distinct life cycle stages indicates there are conserved protein expression between sporozoites and merozoites (Florens et al. 2002). In Inoue et al. mice were immunized with P. yoelii merozoites, treated with mefloquine (MF) for five days post immunization then challenged three weeks post MF treatment with P. yoelii sporozoites resulting in no mice having detectable parasite infection by PCR (Inoue et al. 2012). When reversed, mice immunized with P. yoelii sporozoites, treated with MF and then challenged with P. yoelii merozoites demonstrated a significant reduction in parasitemia levels compared to the controls (Inoue et al. 2012).

The advanced developed malaria vaccine, RTS,S, is a pre-erythrocytic stage vaccine that has been referred to as a leaky pre-erythrocytic vaccine (Moorthy & Ballou, 2009). RTS,S results in partial protection based on blood stage parasites present but at a reduced level. A fully protective vaccine must result no parasite breakthrough. The mathematical model proposed by White and Smith hypothesizes that a multicomponent vaccine may lead to total parasite clearance (White & Smith 2013). Their model delineates how many parasites must be eliminated to have an effective vaccine for both pre-erythrocytic and blood stage vaccines (White & Smith 2013).

The compositions and methods disclosed overcome many of the short comings of previous generation vaccines described above. Vaccine formulations or single component vaccines can be combined to have a synergistic influence to eliminate parasite infection. For example, by combining NYVAC expressing blood stage P. falciparum proteins with the NYVAC expressing pre-erythrocytic stage P. falciparum proteins, a single malaria vaccine formulation expressing 25 P. falciparum antigens termed the malaria SWARM vaccine was created exhibiting a synergistic immunological effect. Specifically, the current invention is superior due to (i) genetic modifications to include codon optimized heterologous genes coupled with synthetic promoters for strong protein expression throughout the virus life cycle and (ii) a reworked vaccine regiment with the possibility of purified proteins as a prime boost strategy.

An effective and long-lasting vaccine towards Pv is desirable to maintain and/or increase the medical readiness of the warfighter deployed or assigned to P. vivax prevalent areas such as South America, India, and Southeast Asia. This NYVAC-based Pv vaccine will also be beneficial to global travelers and citizens visiting or living in P. vivax endemic areas. It has been discovered that it is possible to construct attenuated recombinant vaccinia virus vaccine against Pv (WRPvrNYVAC) containing:

-   -   (1) the Pv circumsporozoite (CS) gene variant VK210 in the J2R         open reading frame;     -   (2) the Pv CS gene variant VK247 in the A56R open reading frame;     -   (3) the Pv apical merozoite antigen 1 (AMA1) gene in the B13/B14         open reading frame;     -   (4) the Pv thrombospodin-related anonymous related protein         (TRAP)/sporozoite surface protein 2 (SSP2) gene in the A26L open         reading frame;     -   (5) the Pv merozoite surface protein 1 [42 kD protein fragment]         (MSP1 fragment p42) gene in the 14L open reading frame;     -   (6) the Pv Pvs25 gene in the C7L-K1L open reading frame region;     -   (7) the Pv Pvs28 gene in the C7L-K1L open reading frame region;     -   (8) the Pv Duffy Binding Protein [region II] (DBP region II)         gene in the C7L-K1L open reading frame region; and     -   (9) one WRPvrNYVAC containing Pv CS gene variants VK210 and         VK247, Pv AMA1, Pv TRAP/SSP2, Pv MSP1 fragment p42, Pv Pvs25, Pv         Pvs28, and Pv DBP region II.         The vaccine can have dual use as a malaria and a smallpox         vaccine. The recombinant NYVAC virus serves as a delivery vector         for the P. vivax proteins to vaccinate against P. vivax. The         NYVAX virus vector also protects against an orthopox virus         infection such as variola virus and monkey pox, because NYVAC is         an attenuated vaccinia virus. Vaccinia virus has been used to         vaccinate against a smallpox infection. Having this dual role is         favorable because there are at least two generations of humans         who are currently vulnerable to a smallpox infection         (unvaccinated). Additionally, certain agents, such as a variola         infection has a mortality rate of approximately 30% and is         identified as a potential bioterrorist agent.

What was newly discovered and unappreciated by Tine et al. and in their patent is that skin scarification (SS) is necessary as part of the method of administering the rNYVAC and in a specific order in which to obtain the best immune response as represented below:

Antibody TNF-α IFN-γ Response Response Response Single IM + − − Single SS − − − SS→SS − + − IM→IM + + − SS→IM + + + IM→SS + + − The “−” indicated no detectable immune response whereas “+” is the appropriate response. Only SS followed by an IM boost produced an antibody response, a TN-Fα response and an IFN-γ response. Tine et al. (1996) failed to appreciate the relevance of the order of skin scarification followed by an intramuscular boost for providing protection to the vaccinated individual. That is at least one reason why their formulation failed to be protective.

In essence, the P. vivax vaccine exemplified combines up to 8 individual recombinant NYVAC each expressing up to 8 unique Pv proteins: 3 (i.e., CS-VK210, CS-VK247, TRAP/SSP2) from the pre-erythrocytic stage, 3 (i.e., AMA1, MSP1 fragment p42, and Duffy Binding Protein region II) from the blood-stage, and 2 (i.e., Pvs28 and Pvs25) from the gametocyte stage for transmission blocking between humans by mosquitoes. However, other vaccines can include only one protein from each stage of Plasmodium life cycle up to all genes, and every gene number in between.

Described herein is a secondary shuttle plasmid for the C7L-K1L region that contains up to 3 multiple cloning sites separated by two native vaccine virus intergenic regions to separate the open reading frames (ORFs). The shuttle plasmids can allow the cloning of 3 distinct P. vivax or P. falciparum malaria genes and place them into the C7L-K1L region in within the NYVAC genome. The recombinant malaria gene containing NYVAC construct or the virion particles produced can be administered as a single vaccine formulation. This can be administered as a single vaccine formulation. The concept of administering a combination of NYVAC constructs containing different protein sequences is termed a WRAIR malaria “SWARM” vaccine.

The vaccine can be administered via different routes. Exemplary administration routes include skin scarification and intramuscularly (“IM”), for example priming the skin by scarification for first vaccination and boost by intramuscular injection. All Pv genes inserted into the WRPvrNYVAC genome are full length. If applicable, the GPI anchor of the malaria protein is removed from the Pv gene before insertion. If applicable, Pv genes with transcription termination sequence can be altered to prevent premature transcription termination. For example, not all native malaria genes contain the vaccinia virus transcription termination sequence TTTTTNT; if this sequence is present, then silent mutations will be introduced that would negatively disrupt the genetic sequence but not the protein codon. Each Pv gene is under the control of a compact synthetic vaccinia virus early/late promoter followed by Kozak's sequence then the start of the Pv gene. Each Pv gene is inserted in the correct orientation within the genome (i.e. operably linked) to minimally disrupt the production messenger RNA (mRNA). A Pv gene is inserted into a WRPvrNYVAC locus to minimally disrupt the production of mRNA. The purpose of this vaccine formulation is to maintain readiness of the military warfighter by preventing disease and non-battlefield injuries associated with malaria.

Two individual WRPvrNYVAC have been constructed to express the full-length protein of Pv CS gene variants, VK210 and VK247. The CS protein expressed from WRPvrNYVAC will be identical to the proteins expressed by the Pv parasite. FIG. 7A-7B demonstrate the synthesis of protein from the WRPvrNYVAC expressed in either CS-VK210 or CS-VK247 in baby hamster kidney-21 (BHK-21) and primary human dermal fibroblast cultured cells. VMP001 consists of a single truncated protein with a chimeric amino acid sequence of both VK210 and VK247 that is expressed as a soluble protein.

Protein expression of P. vivax CS proteins from WRPvrNYVAC is continuous over time. Based on studies performed in multiple human primary cultured skin cells with NYVAC expressing Pf CS protein, the levels of protein synthesis occurred for at least 96 hours. FIG. 8B demonstrates protein translation over 4 days in cultured human dermal fibroblasts for WRPvrNYVAC expressing CS-VK210 and CS-VK247 with corresponding replication titer compared to input virus (FIG. 8A). VMP001, is a soluble protein-based vaccine (Yadava et al., “novel chimeric Plasmodium vivax circumsporozoite protein induces biologically functional antibodies that recognize both VK210 and VK247 sporozoites,” Infect. Immun. 75(3): 1177-85, 2006). The vaccine will immediately deplete over time once administered. Unfortunately, VMP0001, which is a P. vivax vaccine, was used in Phase I/IIa clinical trials that was observed to have a 0.0% efficacy rate when immunized volunteers were challenged with mosquitoes containing live P. vivax parasites.

The NYVAC-based Pv vaccine will reduce the number of doses and time to receive each dose compared to soluble protein-based vaccines. When a vaccinia virus is administered in the form of a smallpox vaccine, after a single vaccination, the estimated protective efficacy is greater than 90% lasting for decades. The leading soluble protein-based malaria vaccine is RTS,S (or RTS,S, tradename Mosquirix™), which targets the Pf CS protein. The most recent clinical trial with RTS,S demonstrated over 80% of volunteers were protected against a Pf malaria infection when given three injections of RTS,S over six months and over 60% of volunteers were protected when given three injections of RTS,S over three months. The clinical trials results with VMP001 demonstrated 0.0% of the volunteers were protected against a Pv malaria infection. In a trial, volunteers received three injections of VMP001 over three months.

The recombinant NYVAC vaccines containing the Pv CS gene variants can be lyophilized and are very stable, not requiring cold storage.

No malaria vaccine currently formulated possesses the features of the constructs provided herein. The NYVAC-based P. vivax vaccine contains CS gene variants VK210 and VK247, AMA1, TRAP/SSP2, MSP1 fragment p42, Pvs25, Pvs28, and DBP region II. The expressed proteins are full length and are continuously expressed. The NYVAC-based P. vivax vaccine does not require an adjuvant, because NYVAC production of its own double-stranded RNA serves as an adjuvant. Pv proteins expressed from NYVAC will be continuously expressed. NYVAC does not require copious amounts of protein to be an effective vaccine as demonstrated by previous smallpox vaccines. This is in contrast, for example, to VMP001, which at the highest dose required 60 μg of the Pv protein. The proteins expressed from NYVAC can produce multiple permutations of the protein based on posttranslational modifications.

Another SWARM vaccine formulation provided here combines up to 25 individual recombinant NYVAC (rNYVAC) vectors, each individual NYVAC vector expressing 25 unique P. falciparum (Pf) proteins (11 from the pre-erythrocytic (liver) stage, 10 from the blood-stage, and 4 from the transmission-blocking (mosquito) stage) can be administered as a single vaccine formulation. The vaccine formulation must have at least one gene from each stage of the malaria parasite life cycle.

The use of a synthetic early/late promoter within recombinant NYVAC developed at WRAIR (WRrNYVAC) generates a greater amount of protein than NYVAC-Pf-7. NYVAC-Pf-7 is a candidate that contains genes encoding seven P. falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle. See Tine, J. et al., “NYVAC-Pf-7: a Poxvirus-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria,” Infect. Immunity, September 1996, p. 3833-3844 (1996). Higher protein titers were obtained with WRrNYVAC (i.e., WRrNYVAC—1×10⁹ pfu; NYVAC-Pf-7 1×10⁷ pfu). Preferable to NYVAC-Pf-7, the constructs disclosed here reduce the overall cost (reagents, supplies, and labor) and the overall amount of vaccine that needs to be administered. Each WRrNYVAC can be easily manufactured in comparison to NYVAC-Pf-7. The WRrNYVAC vaccine formulation can be administered without adjuvant, as the construct produces its own adjuvant in effect. The WRrNYVAC vaccine is stable and can be lyophilized, and hence does not require refrigeration for storage, including long-term storage and dissemination of the formulation in the field or in areas that lack adequate refrigeration.

Current malaria vaccine candidates address only one stage of proteins expressed in the P. falciparum life cycle. A SWARM vaccine addresses all three major environments in which the malaria parasite lives (pre-erythrocytic, blood-stage, and transmission blocking) by being capable of producing at least one gene from each stage of Plasmodium life cycle. The WRAIR malaria SWARM recombinant NYVAC viruses can express any one or more of (as a combination of individual WRrNYVAC vaccines each containing a different protein sequence): 11 pre-erythrocytic proteins, 10 blood-stage proteins, and 4 transmission-blocking proteins. On the other hand, NYVAC-Pf-7 expresses 4 pre-erythrocytic proteins, 2 blood-stage proteins, and 1 transmission-blocking protein but was not effective as administered.

The recombinant NYVAC viruses expressing Plasmodium genes when administered in a vaccine formulation do not require an adjuvant because NYVAC produces its own adjuvant, double-stranded RNA. NYVAC does not require multiple-immunizations because of its ability to continuously generate protein. NYVAC does not require copious amounts to be an effective vaccine as demonstrated by previous smallpox vaccines. Proteins expressed from NYVAC can produce multiple permutations of the protein based on posttranslational modifications. Preferable to what is known in the art; this can provide the immune system a broader scope of epitopes for recognition.

Individuals immunized with a SWARM vaccine also possess added protection against smallpox and other pox viruses. Within the United States, several generations (i.e., children born after 1980) failed to receive a smallpox vaccination, making them vulnerable to smallpox, especially if smallpox were to be weaponized and used as a potential bioterrorist agent. NYVAC can accommodate up to 25,000 base pairs of foreign DNA. Several genes from multiple infectious diseases can be placed within a single NYVAC genome. Furthermore, NYVAC can be easily administered through a skin scarification, a typical method of administering a small pox vaccine formulation. Each full-length P. falciparum gene is controlled by a synthetic early/late promoter.

Each full-length Pv gene is controlled by a synthetic early/late promoter. An exemplary NYVAC vector can contain up to 8 individual recombinant NYVAC vectors expressing 8 unique Pv proteins and administering as one vaccine formulation.

Another SWARM vaccine formulation can combine up to 25 individual recombinant NYVAC (rNYVAC) vectors, each individual NYVAC vector expressing 25 or even 30 unique P. falciparum (Pf) proteins (11 from the pre-erythrocytic (liver) stage, 10 from the blood-stage, and 4 from the transmission-blocking (mosquito) stage) in a single vaccine formulation.

1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood a person skilled in the art. All publications mentioned herein are incorporated herein by reference. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Principles of Neural Science, 4th ed., Eric R. Kandel, James H. Schwart, Thomas M. Jessell editors, McGraw-Hill/Appleton & Lange: New York, N.Y. (2000).

The term “administering” as used herein, means a WRPvrNYVAC vaccine formulation or a WRPfrNYVAC formulation(s) may be administered or performed using any of the various methods or for delivering a biologically active agent to a cell or to a subject in vivo. Also contemplated are WRrNYVAC constructs that express genes from other Plasmodium sp., such as P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii. P. vivax and P. falciparum are just the two predominant species.

As used herein, the term “malaria antigen” refers to any antigen or fragment thereof. The term antigen or fragment thereof, means any peptide-based sequence that can be recognized by the immune system and/or that stimulates a cell-mediated immune response and/or stimulates the generation of antibodies. According to Scand. J. Immunol. 56: 327-343, 2002, considering the whole parasite life cycle, there are essentially three life cycle stages and six targets for a malaria vaccine: (1) sporozoites; (2) liver stages; (3) merozoites; (4) infected RBC; (5) parasite toxins; and (6) sexual stages.

The term “NYVAC” is defined herein as a highly attenuated vaccinia virus strain, NYVAC (vP866), and was derived from a plaque-cloned isolate of the Copenhagen vaccine strain by the precise deletion of 18 open reading frames (ORFs) from the viral genome. Among the ORFs deleted from NYVAC (vP866) are two genes involved in nucleotide metabolism, the thymidine kinase (ORF J2R) and the large subunit of the ribonucleotide reductase (ORF 14L); the gene encoding the viral hemagglutinin (ORF A56R); the remnant (ORF A26L) of a highly expressed gene responsible for the formation of A-type inclusion bodies; the disrupted gene (ORFs B13R/B14R) normally encoding a serine protease inhibitor; and a block of 12 ORFs bounded by two known viral host range regulatory functions (ORFs C7L through K1L). Within this block a secretory protein (ORF N1L) implicated in viral virulence and a functional complement 4b binding protein (ORF C3L) are encoded. The ORFs were deleted in a manner that prevents the synthesis of undesirable novel gene products. The attenuation characteristics of the derived NYVAC strain were compared in in vitro and in vivo studies with those of the Western Reserve (WR) laboratory strain, the New York City Board of Health vaccine strain (Wyeth), the parental plaque-cloned isolate (VC-2) of the Copenhagen vaccine strain used to derive NYVAC, and the avipox virus canarypox (ALVAC), which is naturally restricted for replication to avian species. The NYVAC strain was demonstrated to be highly attenuated by the following criteria: (a) no detectable induration or ulceration at the site of inoculation on rabbit skin; (b) rapid clearance of infectious virus from the intradermal site of inoculation on rabbit skin; (c) absence of testicular inflammation in nude mice; (d) greatly reduced virulence as demonstrated by the results of intracranial challenge of both 3-week-old or newborn mice; (e) greatly reduced pathogenicity and failure to disseminate in immunodeficient (e.g., nude or cyclophosphamide treated) mice; and (f) dramatically reduced ability to replicate on a variety of human tissue culture cells. Despite these highly attenuated characteristics, the NYVAC strain, as a vector, retains the ability to induce strong immune responses to extrinsic antigens. The term “NYVAC-Pf-7” refers to a composition that expresses 4 pre-erythrocytic proteins, 2 blood-stage proteins, and 1 transmission-blocking protein. See “Tartaglia, J., et al., “NYVAC: a highly attenuated strain of vaccinia virus,” Virology, 1992 May; 188(1): 271-232; Tine, J. A., et al. “NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria,” Infect. Immun. 9: 3833-3844 (1996).

The terms “subject,” “host,” and “patient,” as used herein, are used interchangeably and mean an animal being treated with the present compositions, including, but not limited to, simians, humans, avians, felines, canines, equines, rodents, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets. The benefit of vaccinating subjects other than humans is to not only protect animal populations, but to prevent them from being infected source(s) of malaria.

As used herein, a “therapeutic agent” means a compound or molecule capable of producing a therapeutic effect.

As used herein, “therapeutically effective amount” or “therapeutic effect” means an amount of the therapeutic agent sufficient to treat a subject afflicted with a disease (e.g., malaria or poxvirus) or to alleviate a symptom or a complication associated with the disease.

The term “treating” as used herein, means slowing, stopping or reversing the progression of a disease, particularly malaria or poxvirus. As used herein, the terms “treatment,” “treating,” and the like, as used herein refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a condition or disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition or disease and/or adverse effect attributable to the condition or disease. “Treatment” includes any treatment of a condition or disease in a mammal, particularly in a human, and includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression of the condition or disease or symptom thereof.

The term, “Vaccinia virus (VACV)” as used herein means a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, which encodes approximately 250 genes.

The term, “vaccine” or “vaccine formulation” as referred herein, is defined as a pharmaceutical or therapeutic composition used to inoculate a subject in order to immunize the subject against infection by an organism, such as malaria or smallpox. Vaccine formulations typically comprise one or more antigens derived from one or more organisms which on administration to an animal will stimulate active immunity and protect that animal against infection with these or related pathogenic organisms. The vaccine formulation may further comprise an adjuvant, a carrier, an excipient, and/or a stabilizer. The vaccine formulation may be lyophilized and resuspended for administration.

2. Abbreviations

-   AMA-1 apical membrane antigen 1 -   BHK baby hamster kidney cells -   CelTOS cell-traversal protein for ookinetes and sporozoites protein -   CSP circumsporozoite protein -   DBP, RII Duffy Binding Protein Region II -   DNA deoxyribonucleic acid -   EBA RII erythrocyte binding antigen region II -   hDF human dermal fibroblasts -   hDMEC human dermal microvascular endothelial cells -   hK human keratinocytes -   hSK human skeletal muscle -   IFN-γ interferon gamma -   IM intramuscular -   kpb kilo base pairs -   kD kilo Dalton -   LSA1-RPTLS Liver Stage Antigen 1 repeatless -   MOI multiplicity of infection -   MSP1DBL Merozoite surface protein, duffy binding like 1 -   MSP1-p19 merozoite surface protein 1, p19 a proteolytically cleaved     portion of MSP1 -   MSP1-p42 merozoite surface protein 1, p42 is the matured C-terminal     fragment of MSP1 -   NYVAC genetically engineered and attenuated strain deposited with     ATCC under Accession No. Vr-2559 -   ORF open reading frame -   Pf P. falciparum -   Pfs16 Pfs16 surface antigen of P. falciparum -   Pfs28 28 kDa ookinete surface protein of P. falciparum -   Pfs48.45 a sexual state specific malaria antigen -   PfSEA P. falciparum schizont egress antigen-1, or PfSEA-1 -   pfu plaque forming unit -   Pv P. vivax -   Rh5 invasion protein Rh5 (or RH5) -   RNA ribonucleic acid -   rNYVAC recombinant NYVAC -   SIAP1 sporozoite invasion-associated protein 1 -   SIAP2 sporozoite invasion-associated protein 2 -   SPECT1 sporozoite microneme protein essential for cell traversal 1 -   SPECT2 sporozoite microneme protein essential for cell traversal 2 -   SPATR secreted protein with altered thrombospondin repeat -   STARP sporozoite threonine-asparagine-rich protein -   TNF-α tumor necrosis factor alpha -   VACV Vaccinia virus -   WRPvrNYVAC WRAIR Pv recombinant NYVAC may also be seen as WRPvNYVAC -   WRPfrNYVAC WRAIR Pf recombinant NYVAC may also be seen as WRPfNYVAC

3. Constructs

A combination of up to 8 individual recombinant NYVAC expressing 8 unique Pv proteins administered as one vaccine formulation has been shown here to be effective in immunizing a subject and preventing disease and clinical symptoms associated with or caused by both malaria and poxvirus infection. These immunogenic compositions and vaccine formulations can provide for a global vaccine protecting the recipient from disease caused by any strain from any part of the world.

A. Methods of Making a Multi-Stage, Multi-Antigenic Recombinant NYVAC Viral Vector (WRPvrNYVAC) and Uses Thereof

Disclosed is a method for making WRPvrNYVAC viral vector that is multi-stage and multi-antigenic. Methods are provided where an expression cassette is generated that is comprised of synthesized DNA copies of one or more genes as listed below in Table 1 that encode up to 8 multistage P. vivax antigens. The gene is under the tight control of a poxvirus promoter.

TABLE 1 List of P. vivax genes inserted into NYVAC and status of recombinant NYVAC. Protein Recombinant Purification Resolved DNA sequence Expression NYVAC IVR Rounds WRPvrNYVAC confirmed Confirmed CS-VK210 X X X X X CS-VK247 X X X X X TRAP/SSP2 X X AMA1 X X MSP1-p42 X X DBP, RII X X Pvs25 X X Pvs28 X X “X” means that the particular section of the rNYVAC confirmed.

In certain aspects, the gene is under the tight control of a poxvirus promoter. The promoter can be a compact synthetic early-late promoter having a 40-nucleotide sequence identified as SEQ ID NO: 1: AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA. A person of skill in the art could use a naturally occurring endogenous poxvirus promoter. The expression cassette is sub-cloned into a NYVAC donor plasmid to create a shuttle plasmid specific for loci in the NYVAC genome. The shuttle plasmid can be inserted into the A26L, A56R, 14L, J2R, B13/B14R, or C7L-K1L loci within the NYVAC genome (see e.g., FIGS. 9 and 10). After transfecting the shuttle plasmid and simultaneously co-infecting with parental NYVAC to promote in vivo recombination, the recombinant NYVAC viral vector, or WRPvrNYVAC, is generated in an effort to elicit immunity directed against Pv proteins from multiple stages in a malarial life cycle.

B. WRPvrNYVAC

WRPvrNYVAC made according to the methods described herein and as described in the Examples comprises (i) a NYVAC donor plasmid; (ii) a left recombination arm comprising approximately 500 base pairs (bp) upstream of an open reading frame of the NYVAC specific locus placed in the donor plasmid; (iii) a right recombination arm comprising approximately 500 base pairs downstream of the open reading frame of the NYVAC specific locus placed in the donor plasmid; (iv) a compact synthetic vaccinia virus early/late promoter with Kozak's sequence is inserted immediately 5′ the Pv genes; (v) the promoter/gene sequence is inserted between the between the left recombination arm and the right recombination arm that is selected from the group consisting of 8 multistage P. vivax genes set forth in Table 1 above.

Methods for immunizing a subject against malaria and poxviruses are provided. The method comprises administering to the subject an effective amount of the vaccine formulation described herein. Methods are also provided for eliciting a protective immune response in a human against malaria or poxvirus infection comprising administering the vaccine formulation described herein to a human. Administration can be via a route selected from the group consisting of scarification, intradermal injection, subcutaneous (SC) injection, intravenous injection (IV), oral, or intranasal. Administration is preferably via scarification and/or intramuscularly (IM) in order for poxvirus protection to be more effective in the subject.

Methods are provided for treating malaria and smallpox in a subject, comprising administering an effective amount of the vaccine formulation described herein to the subject in need thereof. Other methods contemplate treating malaria and smallpox in a subject by administering a priming vaccine formulation comprising at least one first malaria antigen; and a boosting vaccine formulation comprising at least one second malaria antigen to the subject in need thereof, wherein at least one of the formulations comprises WRPvrNYVAC as described herein. The vaccine formulation protects against disease prior to malaria exposure and infection. The vaccine formulation may alleviate disease and clinical symptoms associated with malaria following malaria or smallpox exposure. The formulation is suitable for rapid immunization with the potential to break the cycle of viral transmission at the individual and population levels.

C. A NYVAC-Based Pv (P. vivax) Malaria Vaccine

Obstacles for effective malaria vaccines include: (i) developmental regulation of antigen expression during parasite replication, (ii) non-responsiveness of individuals to particular parasite antigens or epitopes, and (iii) variability of antigens among different parasite isolates. Vaccine formulations based on single malarial antigens often fail to protect an individual from infection because of these factors. See Tine, J., “NYVAC-Pf7: a Poxvirus-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria,” Infect. Immunity, 64(9): 3833-44 (1996).

Described here is a vaccine formulation that is multi-antigen, i.e., multiple antigens from different stages of the parasite life cycle, with the use of vaccinia virus vectors made from a purified and highly attenuated NYVAC vaccinia virus strain. NYVAC is a highly attenuated strain of vaccinia virus derived from the Copenhagen vaccine by the precise deletion of 18 open reading frames, some of which are associated with virulence or host range. See Tartaglia, J., M. E., et al., 1992, “NYVAC: a highly attenuated strain of vaccinia virus,” Virology 188: 217-232. Once a NYVAC is obtained, it can be used to prepare compositions, such as vaccines, that are effective to generate a prophylactic immune response against malaria infection. Additional embodiments are contemplated wherein the compositions, including vaccines, are effective to generate a therapeutic immune response against malaria infection. A vaccine formulation comprises WRPvrNYVAC and can also serve as a preventive treatment for a poxvirus infection.

A vaccine formulation comprising the WRPvrNYVAC vector is provided for use in the prevention or treatment of malaria and poxvirus, comprising a plurality of malaria-derived antigens. The vaccine composition may include P. vivax malaria antigens selected from the pre-erythrocytic stage include: CS-VK210, CS-VK247, and TRAP/SSP2. The vaccine composition may also include P. vivax malaria antigens selected from the blood stage of the parasitic lifecycle, such as: AMA1, MSP1 fragment p42, and DBP region II. The vaccine formulation may further include P. vivax malaria antigens selected for transmission blocking stage, such as: Pvs25, and Pv28.

Vaccine formulations of the present invention can be tested for sterility, protein, antigen, and nucleic acid content using established assays. For example, residual infectivity can be assayed by inoculation of approximately 5% of the lot volume onto Vero cell cultures, or another suitable cell line, followed by incubation for a sufficient time to amplify any residual infectious virus present. The presence of virus can then be detected by IFA directly on the cells or by plaque assay of the culture supernatants.

The vaccine formulations can be mixed with suitable excipients and/or stabilizers and stored frozen (e.g., −20° C. to −80° C. prior to formulation). The vaccine may be diluted to a titer that is suitable for an immunizing dose in a subject (e.g., a mammal such as a human). The final, vialed vaccine may be tested for purity, identity, osmolality, endotoxin, and sterility by various, standardized assays generally known in the art. The vaccine formulation can also be lyophilized under standard conditions and then stored at 4 to 20° C. and even at higher temperatures (e.g. 40 to 50° C.). Once lyophilized, the vaccine is stable.

Immunogenic potency of a vaccine formulation can be tested by administering the vaccine formulation to mice. An animal efficacy study is designed to demonstrate that the vaccine induces an effective immune response including virus neutralizing antibodies and protection against a live virus challenge in comparison to a placebo control. Additionally, the mouse models are observed during the course of the study for any adverse effects. This testing is necessary before a vaccine can progress to a clinical trial. Typically, such experiments are best performed in a non-human primate infection model (e.g., rhesus macaques) with the primary endpoints being the measurement of virus neutralizing antibodies after vaccination. Protection can be assessed by a disease surrogate such as circulating virus (viremia) after virus challenge. Various vaccine doses and immunization schedules can also be tested in the experiment. Responses can be compared and contrasted for individual animals and among groups using standard statistical methods. For example, log-transformed antibody and viremia titers can be statistically analyzed by ANOVA. Fisher's exact test can be used to compare rates of seroconversion to each virus antigen and viremia rates among vaccine groups and placebo controls. An one-way analysis of variance with a contrast test for trend may be used to assess differences in antibody or viremia titers among groups. To stabilize the variance the analysis is conducted on the logs of the quantified responses. A test for trend using the logistic model can be used to assess differences in the proportion of seroconverts.

Mouse models can be employed for immunological and vaccine formulation effectiveness experiments to optimize the immunization regimen and dose. The vaccine formulation regimen that generates a significant immune response compared to the controls will be the standard regimen implemented for future animal studies. As an example, pre-clinical studies performed in with Modified Virus Ankara (MVA) as a vaccine provides empirical evidence that an immunization strategy that generates the greatest cellular and humoral immune response could be an effective vaccine. Based on the regimen and dose optimization with a single WRPvrNYVAC, it is possible to down select to two viral doses and two vaccine regimens to assess the immune responses when eight (8) rNYVAC constructs are combined in a single immunization formulation (a SWARM vaccine) or in the form of one WRPvrNYVAC construct that simultaneously expresses up to 8 Pv proteins.

Reactogenicity of the vaccine formulations can be monitored and evaluated as may be necessary. A “reactogenicity event” is typically identified as an adverse event that is commonly known to occur for the candidate therapeutic/prophylactic product being studied. Typically, such events are collected in a standard, systematic format using a graded scale based on functional assessment or magnitude of reaction. This provides a risk profile of the candidate product and a defined listing of expected (or unexpected) adverse events, and whether such events are local or systemic events.

The vaccine formulations will be prepared for administration to subjects, in particular, mammals, suitably humans, mice, rats or rabbits, by methods known in the art, which can include filtering to sterilize the solution, diluting the solution, adding an adjuvant and stabilizing the solution. The vaccines disclosed herein may be administered to a human or animal by a number of routes, including but not limited to, for example, parenterally (e.g. intramuscularly, transdermally), intranasally, orally, topically, or other routes know by one skilled in the art. The term parenteral as used hereinafter includes intravenous (IV), subcutaneous (SC), intradermal, intramuscular (IM), intra-arterial injection, or by infusion techniques. The vaccine may be in the form of a single dose preparation or in multi-dose vials, which can be used for mass vaccination programs. Suitable methods of preparing and using vaccines can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton, Pa., Osol (ed.) (1980) and NEW TRENDS IN DEVELOPMENTS IN VACCINES, Voller et al. (eds.), University Park Press, Baltimore, Md. (1978), incorporated by reference.

An exemplary vaccine formulation can be administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, excipients and/or vehicles. An extra adjuvant is not necessary but can be added because the WRPvrNYVAC constructs produce RNA which itself serves as an adjuvant.

The vaccine formulation can be lyophilized to produce a vaccine against malaria or poxvirus in a dried form for ease in transportation and storage. The vaccine can be associated with chemical moieties that may improve the vaccine's solubility, absorption, biological half-life, etc. Such chemical moieties are disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES (1980).

The vaccine formulation may be stored in a sealed vial, ampule, or similar protective vessel. The vaccines disclosed herein can generally be administered in the form of a spray for intranasal administration, or by nose drops, and inhalants. For lyophilized vaccine formulations, the vaccine is dissolved or suspended in sterilized distilled water before administration. Any inert carrier may be used, such as saline, phosphate buffered saline, or any such carrier in which the vaccine components have suitable solubility.

Vaccine formulations disclosed herein may include a carrier. If in a solution or a liquid aerosol suspension, suitable carriers can include, but are not limited to, salt solution, sucrose solution, or other pharmaceutically acceptable buffer solutions. Aerosol solutions may further comprise a surfactant.

Among the acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution, including saline solutions buffered with phosphate, lactate, Tris, and the like. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium, including, but not limited to, synthetic mono- or di-glycerides. Fatty acids such as oleic acid can also be used in the preparation of injectables.

Injectable preparations (injection ready), for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation are also a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

D. Methods of Making a Multi-Stage, Multi-Antigenic Recombinant Pf NYVAC Viral Vector and Uses Thereof

A method for making a recombinant NYVAC viral vector that is multi-stage and multi-antigenic is also disclosed. Methods are provided where an expression cassette is generated that comprises a genomic or a cDNA copy of one or more genes as listed below in Table 2 that encode up to 25 multistage P. falciparum (Pf) antigens (displayed in Table 2). The gene is under the tight control of a poxvirus promoter.

TABLE 2 List of Plasmodium falciparum genes inserted into NYVAC and status of recombinant NYVAC (WRPfrNYVAC). DNA Protein se- ex- Recom- Purifi- quence pression binant cation Resolved con- con- NYVAC IVR Rounds rNYVAC firmed firmed Pre- AMA1 X X X X X erythro- celTOS X X X X X cytic CS X X X X X LSA1- X X X X X RPTLS SIAP1 X X X X X SIAP2 X X X X X SPATR X X X X X SPECT1 X X X X X SPECT2 X X X X X STARP X X X X X TRAP X X X X X Trans- EBA175 RII X X X X X mission MSP1-p19 X X X X X Block- MSP1-p42 X X X X X ing— MSP1DBL X X X X X Blood MSP3 X X X X X stage MSP5 X X X X X MSP7 X X X X X MSP9 X X X X X PfSEA1 X X X X X Rh5 X X X X X Trans- Pfs16 X X X X X mission Pfs25 X X X X X Blocking Pfs28 X X X X X Pfs48.45 X X X  X* X “X” indicated that the task stands completed for the generation of the recombinant NYVAC. The *indicates that there is an alteration in the promoter during the selection process; however, the Pf protein is expressed and the mutation did not impact performance. For Pfs48.45, a single T (thymine) deletion occurred while selecting and purifying the recombinant NYVAC (WRPfrNYVAC). Protein expression was confirmed by using Plasmodium protein specific antibodies and Western blots. DNA sequence was confirmed against the NCBI sequence.

In certain aspects, the gene is under the tight control of a poxvirus promoter. More specifically, the promoter is a compact synthetic early-late promoter having a 43-nucleotide sequence identified as SEQ ID NO: A1: AAAAATTGAAATTTTATTTTTTTTTTTTGGATATAAATAAAA. The promoter can be used to express all P. vivax and P. plasmodium genes as well as the genes from other Plasmodium sp. A naturally occurring endogenous poxvirus promoter can be used. The expression cassette is sub-cloned into a NYVAC donor plasmid to create a shuttle plasmid. The shuttle plasmid is inserted into sites in the NYVAC donor plasmid genome as depicted in FIG. 9, wherein the sites are selected from the group consisting of: A26L, A56R, 14L, J2R, and B13/B14R. After transfecting and simultaneously co-infecting with parental NYVAC virus to promote in vivo recombination, the recombinant NYVAC viral vector, or WRPfrNYVAC, is generated in an effort to elicit immunity directed against multiple stages in a malarial life cycle.

E. Pf WRrNYVAC

In other aspects, Pf WRrNYVAC (also referred to as WRPfrNYVAC) made according to the methods described herein and as described in the Examples comprises: (i) a NYVAC donor plasmid; (ii) a left recombination arm comprising approximately 500 base pairs upstream of an open reading frame of the NYVAC genome placed in the donor plasmid; (iii) a right recombination arm comprising approximately 500 base pairs downstream of the open reading frame of the NYVAC genome placed in the donor plasmid; and (iv) a gene inserted between the between the left recombination arm and the right recombination arm that is selected from the group consisting of 25 multistage P. falciparum genes set forth in Table 2.

In other aspects, methods are provided for treating malaria and smallpox in a subject, comprising administering an effective amount of the vaccine formulation described herein to the mammal in need thereof. Other methods contemplate treating malaria and smallpox in a mammal, by administering a priming vaccine formulation comprising at least one first malaria antigen; and a boosting vaccine formulation comprising at least one second malaria antigen to the mammal in need thereof, wherein at least one of the formulations comprises WRPfrNYVAC as described herein. Exemplary vaccine formulations can protect against disease prior to malaria exposure and infection. A vaccine formulation may alleviate disease and clinical symptoms associated with malaria following malaria or smallpox exposure. Exemplary vaccine formulations are provided that are suitable for rapid immunization with the potential to break the cycle of viral transmission at the individual and population levels.

F. A Pf Recombinant NYVAC-Based Malaria Vaccine

After decades of malaria vaccine research, several major obstacles to the development of an effective vaccine formulation still exist. These obstacles include: (i) developmental regulation of antigen expression during parasite replication, (ii) non-responsiveness of individuals to particular parasite antigens or epitopes, and (iii) variability of antigens among different parasite isolates. Vaccine formulations based on single malarial antigens often fail to protect an individual from infection because of these factors. See Tine, J., “NYVAC-Pf7: a Poxvirus-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria,” Infection Immunity, 64(9): 3833-3844 (1996) and related U.S. Pat. No. 5,766,597.

A vaccine formulation is described that is multi-antigen, i.e., multiple antigens from different stages of the parasite life cycle, with the use of vaccinia virus vectors made from a purified and highly attenuated NYVAC vaccinia virus strain. NYVAC is a highly attenuated strain of vaccinia virus. See Tartaglia, J., M. E., et al., “NYVAC: a highly attenuated strain of vaccinia virus,” Virology 188: 217-232 (1992). Once NYVAC is obtained, it can be used to prepare compositions, such as vaccines, that are effective to generate a prophylactic immune response against malaria infection. Compositions, including vaccines, are contemplated that are effective to generate a therapeutic immune response against malaria infection. In other aspects, the vaccine formulation comprises WRPfrNYVAC and serves as a treatment for poxviruses.

A vaccine formulation is contemplated comprising the WRPfrNYVAC vector is provided for use in the prevention or treatment of malaria and poxvirus, comprising a plurality of malaria-derived antigens. The plurality of malaria-derived antigens should include at least one antigen from each of the 3 (three) stages of the malaria parasite life cycle. An exemplary vaccine composition may include P. falciparum malaria antigens selected from the pre-erythrocytic stage including AMA1, celTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, and TRAP. The exemplary vaccine composition may also include P. falciparum malaria antigens selected from the blood stage, including EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, and Rh5. The vaccine formulation may further include P. falciparum malaria antigens selected from the transmission blocking stage including Pfs16, Pfs25, Pfs28, and Pfs48.45.

Vaccine formulations may be tested for sterility, protein, antigen, and nucleic acid content using established assays. Residual infectivity can be assayed by inoculation of approximately 5% of the lot volume onto Vero cell cultures, or another suitable cell line, followed by incubation for a sufficient time to amplify any residual infectious virus present, which can then be detected by immunofluorescence assay (IFA) directly on the cells or by plaque assay of the culture supernatants. The vaccines can be mixed with suitable excipients and/or stabilizers and stored frozen (e.g., −20° C. to −80° C. prior to formulation). The vaccine may be diluted to a titer that is suitable for an immunizing dose in a subject (e.g., a mammal such as a human). The final, vialed vaccine may be tested for purity, identity, osmolality, endotoxin, and sterility by various standardized assays generally known in the art.

Immunogenic potency of vaccine formulation can be tested by administering the vaccines to mice. An animal efficacy study is designed to demonstrate that the vaccine induces an effective immune response including virus neutralizing antibodies and protection against a live virus challenge in comparison to a placebo control. Also, the animals are observed during the course of the study for any adverse effects. This testing is necessary before a vaccine can progress to a clinical trial. Typically, such experiments are best performed in a non-human primate infection model (e.g., rhesus macaques) with the primary endpoints being the measurement of virus neutralizing antibodies after vaccination. Protection can be assessed by a disease surrogate such as circulating virus (viremia) after virus challenge. Various vaccine doses and immunization schedules can also be tested in the experiment. Responses can be compared and contrasted for individual animals and among groups using standard statistical methods. For example, log-transformed antibody and viremia titers can be analyzed by ANOVA. Fisher's exact test can be used to compare rates of seroconversion to each virus antigen and viremia rates among vaccine groups and placebo controls. A one-way analysis of variance with a contrast test for trend may be used to assess differences in antibody or viremia titers among groups. To stabilize the variance the analysis is conducted on the logs of the quantified responses. A test for trend using the logistic model can be used to assess differences in the proportion of seroconverts.

Mice can be used for immunological and vaccine formulation effectiveness experiments in search of optimization of immunization regimen and dose. The vaccine formulation regimen that generates a significant immune response compared to the controls will be the standard regimen implemented for future animal studies. Furthermore, as an example, pre-clinical studies performed in with Modified Virus Ankara (MVA) as a vaccine provides empirical evidence that an immunization strategy that generates the greatest cellular and humoral immune response could be an effective vaccine. Based on the regimen and dose optimization with single WRrNYVAC, it is possible to down select to two viral doses and two vaccine regimens to assess the immune responses when 25 rNYVAC are combined as a single immunization (SWARM vaccine).

G. Routes of Administration and Dosage

The preferred routes of administration and dosage regimen may be determined by a physician, and depend on the age, sex, weight, of the subject, and the stage of the disease. Administration may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration is preferably in a “therapeutically effective amount” this being sufficient to show effect or benefit to the subject. It will also depend upon potential toxicity, overall health, and age of the subject. Decisions on administration are within the responsibility of those of skill in the art.

Dosing is dependent upon severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a reduction of symptoms is achieved. Dosing may can also be age dependent. One of skill in the art is knowledgeable about optimal dosing schedules as they can be calculated from measurements of accumulation in the body. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. Therapeutically effective amounts (dosages) may vary depending on the relative potency of individual characteristics and can generally be routinely calculated based on molecular weight and EC50s in in vitro and/or animal studies. For example, an intramuscular (IM) injection generally requires a higher dosage of vaccine as compared to administration via scarification. However, the dose and formulation administered to a subject IM versus skin scarification can be the same. Generally, the amount of rNYVAC particles administered is 10⁶ or 10⁷. If there is more than one rNYVAC being administered in a SWARM formulation (multiple rNYVAC viruses), each virus should be present at the same dosage. Additionally, lower dosages that are therapeutically effective can be dosed for children versus adults.

While single injection of the vaccine can be administered, given the results, administration by skin scarification followed by a IM boost is the preferred order and number of doses. Routes of administration may include a single skin scarification or a single intramuscular injection (IM). Other routes may include a prime single skin scarification and then boost with a single scarification, or by first administering the prime via IM, followed by a boost IM. In other aspects, prime single skin scarification is preferred with an IM boost. Finally, in yet other aspects the route of immunization is prime IM and then a boost of single skin scarification. Less preferred are combinations including subcutaneous administration and intradermal administration of the virion either alone or in other administration combinations.

H. Kits

Kits are provided comprising the vaccine formulation described herein. Vaccine formulations of the present invention may be placed within containers, or kits, along with packaging material, which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.

EXAMPLES

Illustrated below are experiments, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. The Materials and methods used for each of the examples are provided below unless otherwise described.

Materials & Methods for Isolating and Crypreserving Murine (Mouse) Splenocytes:

Autoclaved scissors (2); Autoclaved forceps (2); 15 mL conical tubes; 50 mL conical tubes Pipette man; P-1000 plus tips; 70% Ethanol (ETOH); Absorbent Pads; DMEM or RPMI media; 1 cc syringes; 70 μM cell strainers; Ice Bucket; Freezing Container (Mr. Frosty); Fetal Bovine Serum (FBS), and DMSO.

Prepare Freezing Media (90% FBS+10% DMSO):

-   -   1. Calculate the appropriate amount of freezing media needed for         mouse study and prepare a couple mL excess.     -   2. Thaw FBS and aliquot needed volume into 50 mL conical tube         that is covered with aluminum foil.     -   3. Open new ampule of DMSO and add appropriate amount to FBS and         place in refrigerator until samples have been processed.     -   4. Medium needs to be stored protected from light.         Animals are euthanized and spleens are removed under sterile and         hood conditions. Obtain blood from the animal by cardiac stick.         Transfer blood from syringe to blood collection tube. After         blood has set a room temperature for 15 minutes transfer to ice         bucket. Transfer mouse to biological hood and spray right side         of mouse with 70% ETOH as diagramed in FIG. 1. Remove the mouse         spleen. Transfer spleen to 15 mL conical tube containing 4-5 mL         of DPBS and place on ice.

Splenocyte Isolation and Cryopreservation Procedure:

Setup ice bucket in hood with 50 mL conical tubes. Place sterile 70 μM cell strainer inside 50 mL conical tube that is on ice. Pour contents of mouse spleen into the conical tube containing the cell strainer. Using the back end of the syringe plunger, mash the spleen into the cell strainer. Pipette 10 mL of media to rinse the plunger and filter to ensure mashed spleen is transferred into 50 mL conical tube. Note: Added media may not pass into conical tube immediately due to seal created with cell strainer, carefully lift strainer to allow media to wash contents of strainer into 50 mL conical tube. Place 50 mL conical tubes containing mashed spleen on ice until entire set of spleens are mashed. It is advised to work in groups of 15-20 spleens at a time. Centrifuge set of mashed spleens at 350 g for 5 minutes at 4° C. Aspirate Media and resuspend cell pellet in 1 mL of Freezing Media. Transfer to freezing container. Once all cells have been resuspended in freezing media transferred to a freezing container transfer cells to −80° C. freezer. Do not disturb freezer or container for 24 hrs. After 24 hrs transfer harvested splenocytes to liquid nitrogen tank for long term storage. Cells should be transferred between 24-72 hrs.

Materials and Methods for Mycophenolic Acid (MPA) Selection of Vaccinia Virus:

At two days prior to the selection, Acquire BHK cells in preparation for MPA selection of VV (vaccinia virus) recombinants. Establish the number of controls and the number of dilutions to determine the total amount of cells needed. Recommend at least 3 dilutions (1:10, 1:100, 1:1000) for each virus.

The day prior to the selection, seed approximately 1.5-2×10⁶ BHK cells/60 mm dish using BHK specific media. MPA pretreatment begins between 16-20 hours post seeding to allow the BHK cell monolayer to reach ≥70% confluency. Alternatively, one can seed 3×10⁶ BHK cells/60 mm dish 3 hrs prior to beginning MPA selection using BHK media.

On the day of selection, prepare fresh MPA solution per 50 mL of BHK media as follows: 75 μL Hypoxanthine (10 mg/mL) Sigma: 9636; 50 μL Mycophenolic Acid (MPA, 10 mg/mL, Sigma: M3536); and 1.25 mL Xanthine (10 mg/mL, Sigma: X7375).

Pretreat the BHK cells by waiting for the cells to adhere to the bottom of the dish, add 4 mL of BHK media and the fresh MPA solution to each 60 mm dish that will be used for selection. Pretreat the cells for at least 6 hours. Do not exceed 8 hours of culturing for the BHK cells, otherwise the cells will die. Prepare dilutions of virus IVR solution or X round of +MPA selection.

Prepare 3 sets of dishes for each virus selection. Use dilutions (1) 1:10, (2) 1:100, (3) 1:1000. Have enough dilution volume to infect with 100 uL of virus dilution.

Infecting the BHK cells is performed by labeling dishes—Initials, date, VV recombinant, MPA round, dilution. Aspirate MPA solution from the BHK monolayer. Add 100 uL virus dilution solution to each specified 60 mm dish. Rock the 60 mm dish every 10 minutes (for 1 hour) always keeping the entire monolayer hydrated. In between rocking, place the infected 60 mm dishes in 37° C., 5% CO₂ incubator. After 1 hour of rocking, add 4 mL of BHK media and MPA solution to each 60 mm dish. Incubate the cells for 24-36 hrs at 37° C., 5% CO₂.

The following day (Day +1), check for plaques & adding agarose overlay. It is hard to distinguish plaques in the BHK cell line with your eye, so check for plaques using a light microscope. Once you see plaques, overlay the BHK monolayer with Low Melt Agarose solution+MPA+X-gal. Ingredients: 25 mL 3% Agarose, 25 mL 2×MEM, 500 μL X-gal (20 mg/mL), MPA solution (see above). This will make a 50 mL solution.

Aspirate BHK media from the monolayer. Add 4 mL of “37° C.-45° C.” overlay agarose solution to each 60 mm dish. Allow the agarose to solidify by leaving plates on a counter top at room temperature for 15 minutes. Then place back into the incubator. Incubate for 6-20 hours at 37° C., 5% CO₂ to allow expression of blue plaques. You are looking for blue plaques based on the 1^(st) round recombination event.

On Day +2, at this point there will be blue colored plaques. As of now, these plaques are the desired recombinants. Circle 5 distinct blue plaques that are not in close proximity to white plaques. If both types of plaques are picked, this will give a mixed population of viruses. Core out each circled blue plaque using a Pasteur pipette. For example, deposit each of the 5 blue plaques into a microfuge tube containing 200 uL of 1×MEM, 2% FBS. One can also keep the plaques separate and select individually, and pool at the end. Proceed to freeze thaw protocol.

On Day +3 of selection, to ensure there is no mixed population of recombined/not recombined viruses, 2 more rounds of +MPA selection will have to be performed to select for distinct blue plaques. Once 3 rounds of +MPA selection are performed, it will be time to initiate the 2^(nd) recombination event and resolve the virus for the desired recombinant. For the next 3 rounds there will be no MPA used. This will be like a normal infection as follows.

At 2 days prior to selection, acquire RK cells (or BHK if your particular virus does not replicate in RK cells) in preparation for −MPA selection of VV recombinants. Establish the number of controls and the number of dilutions to determine the total amount of cells needed. Recommend at least 3 dilutions (1:10, 1:100, 1:1000) for each virus.

One day prior to selection, seed approximately 1.5-2×10⁶ RK cells/60 mm dish using RK specific media. Allow the RK cell monolayer to reach ≥70% confluency. Again, as an alternative, one can seed 3×10⁶ RK cells/60 mm dish 4 hrs prior to beginning infection using RK media.

For the day of selection without MPA (−MPA), prepare dilutions of the previous X round of +MPA or virus X round of −MPA selection. As mentioned in Day −2, have three sets of dishes for each virus selection. Use dilutions (1) 1:10, (2) 1:100, (3) 1:1000. Have enough dilution volume to infect with 100 uL of virus dilution. To Infect the RK cells: (a) label dishes—Initials, date, VV recombinant, MPA round, dilution; (b) aspirate RK media solution from the RK monolayer. (c) Add 100 uL virus dilution solution to each specified 60 mm dish. (d) Rock the 60 mm dish every 10 minutes (for 1 hour) always keeping the entire monolayer hydrated. In between rocking, place the infected 60 mm dishes in 37° C., 5% CO₂ incubator. (e) After 1 hour of rocking, add 4 mL of RK only media to each 60 mm dish. (f) Incubate for 24-36 hrs at 37° C., 5% CO₂.

At day +1 selection, check for plaques and add agarose overlay. It is easy to distinguish plaques in the RK cell line by eye, but also check for plaques using a light microscope. Once you see plaques, overlay the RK monolayer with Low Melt Agarose solution+X-gal with no MPA. Ingredients: 25 mL 3% Agarose, 25 mL 2×MEM, 500 μL X-gal (20 mg/mL). This will make a 50 mL solution. Aspirate RK media from the monolayer. Add 4 mL of “37° C.-45° C.” overlay agarose solution to each 60 mm dish. Allow the agarose to solidify by leaving plates on a counter top at room temperature for 15 minutes. Then place back into the incubator. Incubate for 6-24 hours at 37° C., 5% CO₂ to allow expression of blue plaques. You are looking for blue plaques based on the 1^(st) round recombination event.

On day +2 after selection is begun, there will be blue & white plaques. The white plaques are the desired recombinants. Circle 5 distinct white plaques that are not in close proximity to blue plaques. If both types of plaques are picked, this will give a mixed population of viruses. Core out each circled white plaque using a Pasteur pipette. Deposit 5 white plaques into a microfuge tube containing 200 uL of 1×MEM, 2% FBS. Proceed to freeze thaw protocol.

On Day +3 after selection is started, to ensure there is no mixed population of 1^(st) vs. 2^(nd) recombination event viruses, perform 2 more rounds of −MPA selection using the RK cell line will have to be performed to select for distinct white plaques, which are the desired recombinants. A total of 3 rounds of +MPA (BHK cells) and 3 rounds of −MPA (RK cells) should be performed. Proceed to vaccinia virus amplification protocol.

Materials and methods for plasmid in vivo recombination (PIVR): Always perform under aseptic conditions. All PIVR PCR products must be purified by using Qiagen plasmid isolation kit.

At Day −2 (two days before) PIVR, acquire BHK cells in preparation to perform PIVR. Establish the number of controls and the number of recombinants to determine the total amount of cells needed. ALL PIVR reactions should be performed in duplicate.

At Day −1, seed approximately 6-8×10⁵ BHK cells/35 mm dish using BHK specific media. PIVER should be begun between 16-20 hours post seeding to allow the BHK cell monolayer to reach ≥70% confluency.

At Day 0, begin PVR as follows. Prewarm Opti-MEM media (Invitrogen: 31985-070). Use a total of 3 mL for each 35 mm dish. 2 mL of Opti-MEM (−FBS, no FBS) during procedure and 1 mL Opti-MEM (+FBS) for incubation. The steps are formulated to reduce significantly the amount of FBS required for cultivating mammalian cells in vitro. Determine your DNA concentration—NEED 500 ng of plasmid DNA for each PIVR. To determine the amount, use a Nanodrop—OD₂₆₀ UV spectrophotometer. Serial 2-fold dilutions—agarose gel. Check the confluency of the dishes. Proceed with PIVR when cells are 70%-100% confluent. Aspirate BHK specific media off and & add 2 mL of pre-warmed Opti-MEM media to each 35 mm dish. Allow the BHK cells to incubate in Opti-MEM for a minimum of about 30 minutes to a maximum of 3 hrs.

Prepare transfection mix: (The reagents are located in the 4° C. cell culture room refrigerator). Dilute the 500 ng of DNA product with Opti-MEM to a final volume of 100 L. Vortex and aliquot the amount of PLUS Reagent (Invitrogen) needed from the master stock. Add 6 μL of PLUS reagent (Invitrogen: 11514-015) to each DNA/OptiMEM solution.

Adding PLUS reagent enhances cationic-mediated transfection of DNA into cells. Vortex DNA/Opti-MEM/PLUS solution and incubate for 15 minutes at room temperature. After a 15 minute incubation, add 100 uL of Opti-MEM/Lipofectamine (Invitrogen) solution to the DNA mix. Add 6 μL of Lipofectamine® (Invitrogen: 18324-012) plus 94 μL of Opti-MEM to each PIVR. Make a master mix and then aliquot the Opti-MEM/Lipofectamime solution to each PIVR. Gently mix DNA/PLUS/Opti-MEM/Lipofectamine solution and incubate for 15 minutes at room temperature. The final volume should be 200 μL. During incubation time, thaw the virus that will be used for PIVR in the virus room H₂O bath. Calculate the amount VV that will undergo PIVR. This is determined by the approximate number of cells in the 35 mm dish being used for in vivo recombination. Use a virus MOI (multiplicity of infection) of 0.05 (1 pfu/20 cells) for each 35 mm dish. After the 15 minute incubation time, transport all 35 mm dishes to a virus room. In the virus room, add the calculated amount of VV directly to the transfection solution and gently mix.

Infecting the BHK cells: Aspirate Opti-MEM off the BHK monolayer. Add the entire 200 μL transfection mix to each specified 35 mm dish. Rock the 35 mm dish every 10 minutes (for 1 hour) always keeping the entire monolayer hydrated. In between rocking, place the infected 35 mm dishes in 37° C., 5% CO₂ incubator. After 1 hour of rocking, add 1 mL of Opti-MEM, 2% FBS to each 35 mm dish. Incubate for 36-48 hrs at 37° C., 5% CO₂.

At Day +2, harvest. Check dishes for CPE (cytopathic effect/cell rounding), scrape the cells into the media using the blunt end of a P1000 pipet tip or a cell scraper and transfer the −1 mL solution to a microfuge tube. Pellet cells using a swinging bucket rotor at 1,000×g, 4° C. for 10 minutes. Discard supernatant. Resuspend the cell pellet in 200 uL of 1×MEM, 2% FBS. Proceed to freeze thaw protocol.

Vaccinia Virus Genome Isolation Methods and Materials:

Acquire at least 1×10⁸ pfu to isolate the viral genome. A titer less than the one specified can work depending on the prep. Bring volume up to 100 μL using 1 mM Tris at pH 8.8. Add 100 μL of Phenol (Sigma: P4557) at pH >7. Gently invert 5 times to form a white precipitate. Spin at 12,000×g at room temperature for 2 minutes. Remove top layer (aqueous layer) and place in new microfuge tube. Discard bottom layer (organic layer). Add 50 μL Phenol (pH: basic)+50 μL Chloroform:Isoamyl Alcohol (24:1) to the aqueous layer. Gently invert 5 times. Spin at 12,000×g at room temperature for 2 minutes. Remove aqueous layer and place in new microfuge tube. Discard organic layer. Add 100 μL Chloroform:Isoamyl Alcohol (24:1) to the aqueous layer. Gently invert 5 times. Spin at 12,000×g at room temperature for 2 minutes. Remove aqueous layer and place in new microfuge tube. Discard organic layer. Add 20 μL 2.5 M Ammonium Acetate and 250 μL 95% Ethanol to the aqueous layer. Place in freezer (−20° C.) for at least ½ hour to overnight. Spin ≥14,000×g at 4° C. for 20 minutes. Discard supernatant with p1000 pipette. Sometimes a translucent pellet is visible at the bottom of the microfuge tube. Add 500 μL 70% Ethanol to the microfuge tube containing DNA. Briskly shake. Spin ≥14,000×g at room temperature for 10 minutes. Discard supernatant using p1000 pipette. Repeat steps the 70% enthanol steps followed by centrifugation.

Remove the entire remaining supernatant with p200 pipette. Air dry (in a closed drawer) for at least 2 hours to overnight. Alternatively, speed vacuum dry the DNA. Resuspend the DNA with 15 uL of sterile water. Scrape the sides and bottom of the microfuge tube to fully resuspend the DNA. Proceed to vaccinia virus genome PCR protocol.

Example 1. Construction of Shuttle Plasmids and Insertion of Pv Genes

Insertion of Plasmodium genes within the NYVAC genome occurred within the loci of A26L, A56R, 14L, J2R, B13/14R, or C7L-K1L (or C7LKIL), because these gene products are non-essential for vaccinia virus (VACV) replication in cells in culture (Antoine et al., Gene 177: 43-46, 1996; Child et al., Virology 2: 625-9, 1990; Scheiflinger et al., Arch. Virol. 141: 663-69, 1996). Oligonucleotides were generated with unique restriction enzyme sites approximately 500 basepairs upstream and approximately 500 base pairs downstream from the A26L, A56R, 14L, J2R, B13/14R loci and genes in the region of C7L through K1L from NYVAC genomic DNA. The PCR fragments were ligated into the multiple cloning site (MCS) of the pBS SK-plasmid (pBluescript SK, Addgene) as depicted in FIG. 9. After diagnostic restriction enzyme digest to confirm fragment insertion into their respective plasmid, the A26L, A56R, 14L, J2R, and B13/14R loci were displaced and replaced with a multiple cloning site (MCS) by using converging megaprimers via whole plasmid PCR to construct pMPΔA26L:MCS, pMPΔA56R:MCS, pMPΔI4L:MCS, pMPΔJ2R:MCS, and pMPΔB13/14R:MCS shuttle plasmids. The shuttle plasmid for the C7L-K1L region was constructed with 3 distinct MCS in between 2 modified intergenic DNA regions similar to the DNA sequence in vaccinia virus Copenhagen strain. This DNA sequence was synthetically generated and ligated in between the left and right recombination arms for the C7L and KIL region to construct pMPΔC7L-K1L:2IGR, 3MCS shuttle plasmids.

The E. coli gpt gene which encodes the xanthine guanine phosphoribosyl transferase is driven by an entomopox virus promoter and was sub-cloned into all shuttle plasmids. This allowed for selection of recombinant NYVAC containing heterologous genes when in the presence of mycophenolic acid (MPA) (Falkner & Moss 1988).

The Pv genes were identified in National Center Biotechnology Information database. The Pv genes were then annotated with a VACV derived compact, synthetic early/late promoter at the 5′ end (SEQ ID NO: A1). Under normal conditions, the compact synthetic early/late promoter exhibited at least 50 times greater gene expression than the widely used P7.5 early/late (E/L) promoter [Chakrabarti et al., 1997]. The promoter/Pv gene sequences were synthesized by GENEWIZ gene synthesis technology pursuant to our direction. To generate complete plasmids, each Pv gene was sub-cloned into one of the following shuttle plasmids: pMPΔA26L: MCS, pMPΔA56R: MCS, pMPΔI4L: MCS, pMPΔJ2R: MCS, pMPΔB13/14R: MCS, or pMPΔC7L-K1L:2IGR, 3MCS.

Example 2. In Vivo Recombination and Selection of Recombinant NYVAC

In vivo recombination (“IVR”) between wild-type NYVAC (wtNYVAC) and a shuttle plasmid will generate a recombinant NYVAC according to the materials and methods described above.

Confluent baby hamster kidney (BHK-21) cell monolayers were individually transfected with shuttle plasmids containing Plasmodium spp. genes using Lipofectamine®/Plus reagent mixture (Invitrogen) according to manufacturer's instructions. The BHK-21 cell monolayer was subsequently infected with wild type (wt) NYVAC at a low multiplicity of infection (MOI) with slight modifications according to Kibler, K. V., et al., 1997, e.g., 0.5 MOI. The transfected/infected cells were incubated in the presence of antibiotic-free Opti-MEM (Invitrogen) at 37° C. in 5% C02 for 48 hours to allow homologous recombination between shuttle plasmid containing Plasmodium genes and NYVAC. The selection of NYVAC containing integrated shuttle plasmid was accomplished by 3 rounds of MPA (mycophenolic acid) selection on confluent African green monkey cell (BSC-40) monolayer as described above. After 3 rounds of selection for recombinant NYVAC (rNYVAC) in the presence of MPA, the extraneous shuttle plasmid sequence incorporated within the NYVAC genome was removed through a second recombination event while retaining the Plasmodium gene of interest. This required 3 subsequent rounds of infections and plaque isolation in the absence of MPA to acquire purified WRPvrNYVAC. The isolated WRPvrNYVAC plaques were amplified to make stocks and viral titer was determined. An aliquot of the amplified stock (stock concentration is generally about 1e⁷ to 1e⁹ pfu per milliliter) was subjected to phenol/chloroform to extract the rNYVAC genome. The region flanking the newly inserted transgene underwent PCR and DNA sequencing to determine correct insertion. To confirm transgene stability, the rNYVAC underwent at least 10 cell passages under normal cell incubation conditions of 5% CO₂ and 37° C. before then being plated to isolate individual plaques as described above, amplified, and subjected to Western blot analysis to confirm preservation.

To achieve a single WRPvrNYVAC expressing simultaneous Plasmodium proteins, WRPvrNYVAC expressing the first Pv protein served as the parental virus for subsequent Plasmodium gene insertion by IVR the location of insertion will be based on the shuttle plasmid transfected. The WRPvrNYVAC expressing simultaneous P. vivax proteins will be resolved using the MPA selection method. A single WRPvrNYVAC can have up to eight Plasmodium proteins expressed simultaneously.

Example 3. Multi-Cycle Replication Profile of rNYVAC

To assess viral replication, confluent baby hamster kidney cells (BHK-21), human keratinocyte (hK), human dermal fibroblasts (hDF), human dermal microvascular endothelial cells (hMVEC-D), and human skeletal muscle cells (hSM) monolayers will be mock infected, infected with wild-type NYVAC (wtNYVAC), or recombinant NYVAC (rNYVAC) expressing Pv protein at a low multiplicity of infection (MOI) in triplicate for each infection. Virus adsorption will occur on cell monolayers at 37° C. for 1 hour. The monolayer will be washed and incubated with complete cell culture media at 37° C., 5% CO₂ atmosphere. Infected cells will be harvested 2 hours post infection (hpi), 1 day post infection (dpi), 2 dpi, 3 dpi, and 4 dpi. The cells will undergo multiple freeze-thaw cycles to release infectious virus. The viral titer is measured by plaque assay on BSC-40 cells. The 2 hpi serves as the input amount of virus to take into account such factors as virus adherence to plastic ware and the efficiency of adsorption; the 2 hpi serves to measure the true titer of virion that infects the cells. Each dpi (day post infection) will be divided by the input virus titer to determine if the rNYVAC construct was replication competent or replication abortive in each cell line. Once plaques are resolved, the BSC-40 monolayer is stained with crystal violet. Staining is performed by quickly removing the media and adding 1 mL of 0.2% crystal violet stain (0.1% crystal violet in 20% ethanol). Cells cannot be permitted to dry out before adding the stain because this will result in a poor cell monolayer for counting the plaques. Staining is performed for about 1 minutes and then the stain is aspirated off. The plates are gently washed with distilled, deionized (dd H₂O) water. The plate is then allowed to dry, and plaques are subsequently counted. Plaques are counted in wells wherein there are 20-200 plaques.

The data is presented as plaque forming unit (PFU or pfu) per mL of cell lysate. The indicated cell lines will be infected at an MOI of 0.05 with wtNYVAC, or recombinant NYVAC expressing Pv protein. The infected cells will be harvested 2 hpi, 1 dpi, 2 dpi, 3 dpi, and 4 dpi. See FIG. 8A-2B.

Example 4. Evaluation of Pv Protein Expression from WRPvrNYVAC

A second set of dishes will be seeded in parallel to the multi-cycle assays. The cell monolayers are mock infected with wtNYVAC, or recombinant NYVAC (rNYVAC) expressing Pv protein at a low MOI (0.5 MOI of the indicated rNYVAC virus). Infected cells are harvested at the same time as the multi-cycle assays (2 hpi, 1 dpi, 2 dpi, 3 dpi, and 4 dpi and generally carried out in 12-well plates). The cells are lysed in Laemmli buffer, centrifuged through a Qiagen® Qiashredder according to manufacturer's instructions, and cell extracts are fractionated using a 4-15% gradient SDS-PAGE.

Example 5. Protein Expression Corresponding to Multi-Cycle Growth Curves of WRPvrNYVAC

The indicated cell lines will be infected at an MOI of 0.05 with wtNYVAC or recombinant NYVAC (rNYVAC) expressing Pv protein as described above. The infected cells will be harvested and lysed with Laemmli buffer at 2 hpi, 1 dpi, 2 dpi, 3 dpi, and 4 dpi. The whole cell lysates will be fractionated using a denaturing 4-15% gradient SDS-PAGE. Western blot analysis will be performed for the presence of Pv proteins. The blot can be stripped and reprobed for the presence of the cellular protein β-actin. The β-actin protein serves as a loading control between all samples tested.

Example 6. Pv Transgene Stability within rNYVAC Genome

The WRPvrNYVAC will be passaged for 10 passages in the permissive BHK-21 cell line in accordance with the description above. To assess Pv gene sequence fidelity and retention, the WRPvrNYVAC will be used to infect BHK-21 cells at an MOI of 0.05, with experiments performed in duplicate. The rNYVAC in dish 1 will be harvested and used to infect subsequent BHK-21 cells. Cell extracts will be generated from dish 2 to confirm Pv protein expression. Stability experiments will be performed when 8 WRPvrNYVAC are co-infected.

Example 7. Safety Profile of rNYVAC

The intracranial infection of newborn mice developed by the FDA Center of Biologics Evaluation and Research is the most sensitive animal model to determine poxvirus pathogenesis (Li et al. 2004). This method is 100 to 1,000 times more sensitive when assaying viral pathogenicity compared to the severe combined immunodeficient (SCID) mouse model (Vijaysri et al., Vaccine 26: 664-76, 2008).

Pregnant CD-1 mice within 10 days of gestation will be used. Three days post birth, the mouse pups receive a 10 μL intracranial injection of 1.0×10⁰ (1 PFU) up to 1.0×10⁸ PFU at ten pups per virus dose. The newborn mice will be monitored twice a day post intracranial injection for 14 days to assess morbidity and mortality. The pathogenicity of a WRPvrNYVAC is compared to wtNYVAC. VACV-Copenhagen, the parental strain of wtNYVAC, is used as the positive control for pathogenicity in accordance to the methods and materials described in Kibler et al. 2011.

Example 8. Detection of Pv Specific Antibodies

ELISAs will be performed to determine Pv-specific antibodies from serum collected from the mice as described above. The antibody titers will be expressed as the reciprocal of the highest serum dilution that is above baseline value.

Example 9. Detection of NYVAC-Neutralizing Antibodies

Anti-vector immunity can reduce efficacy of the vaccine; therefore, the presence of neutralizing antibodies against VACV will be tested according to the materials and methods as described in Reyes-Sandoval et al., Mol. Ther. 8: 1633-47, 2012. The collected mice serum, collected as described above, will be serial two-fold diluted in minimal essential media (MEM) and mixed with 100 PFU wtNYVAC, and then cultured overnight at 37° C., 5% CO₂ in duplicate. Mock infected mice serum or MEM will be mixed with 100 pfu wtNYVAC to serve as reference controls. Confluent BSC-40 monolayers will be inoculated with the serum-virus mixture and allowed to incubate for virus adsorption for 1 hour at 37° C. at 5% CO₂. After 1 hour incubation, BSC-40 cells will be overlaid with complete MEM and placed in the incubator for 24-48 hours at 37° C. and 5% CO₂. Once plaques are present in reference controls, the monolayers will be stained with crystal violet and plaques are counted. Plaques are manually counted for each serum sample.

The endpoint NYVAC neutralizing antibody titer is defined as the reciprocal of the highest serum dilution serum that results in greater than 50% plaque reduction compared to the virus-MEM control mix.

Example 10. Assessment of Activated Cell Mediated Immunity

The Luminex™ cytokine mouse magnetic or Mesoscale© 10-plex panel (or 10-plex human panel when humanized mice will be tested) will be used to identify active Th1/Th2 immune response through the detection of 10 analytes (i.e., GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p40/p70), and TNF-α; U-PLEX TH1/TH2 Combo Mouse, Cat No. K15071K) that can be harvested spleens or lymph nodes according to manufacturer's instructions. Cells will be cultured, stimulated with escalating amounts (0.1 through 3.0 μg) of Pv-specific peptides for 48 hours. The mouse cohorts included those exposed to wild-type (wt) NYVAC, WRrNYVAC-PfCSP, WR7AS1, and WR25AS1. At Day 1 a cohort of 3-5 mice were administered a dose of 1×10⁶ pfu of each construct (Day 0). They received a boost of the same construct at 25. Mice were euthanized and spleens collected at day 50 after the first administration.

Animals were tested for their weight response to immunization. At the time of harvest, the percent weight change for animals having received the WRrNYVAC-PFCSP was consistently greater after week 1 than the mock, wtNYVAC, WR7AS1, and WR25AS1 treated cohorts.

The splenocytes will be pooled and stimulated with the indicated Pf stimulants (i.e., peptide pools or whole proteins) with various doses (1.0, 3.0, 10 μg for whole proteins) and (0.1, 0.3, 1.0, and 3.0 μg for peptide pools. An ELISA can be performed for Pf-specific antibodies that recognize Pf antigens AMA, CelTOS, LSA, MSP, CSP, and TRAP.

The supernatants are harvested 48 hours post infection and assayed using the Mesoscale V PLEX Proinflammatory Panel 1 Mouse Kit on the MESO QuickPlex SQ 120 instrument according to manufacturer's instructions.

Example 11. Pv Challenge in Humanized Mice

We will test the WRPvrNYVAC vaccine effectiveness against a P. vivax challenge through humanized mice. Based on the optimization dose and vaccine regimen studies in mice with recombinant NYVAC expressing P. falciparum proteins we will apply this information to perform vaccine efficacy studies of recombinant NYVAC (rNYVAC) expressing Pv proteins in humanized mice against live P. vivax malaria parasites. Briefly, groups of 5 animals (DRAGA mice) will receive the first immunization on Day 01 and a second immunization 25 days later. On Day 50, the immunized mice will be challenged by mosquito bite (up to 15 mosquitoes per animal containing Pv parasites). After mosquito bite, the mosquitoes will be collected, dissected, and the glands will be evaluated for the presence of Pv parasites. Days 7, 14, and 21 days post mosquito bite challenge, mice venous tail blood will be collected to monitor for the presence of Pv parasites, Pv through employing the highly sensitive Malaria Multiplex Sample Ready PCR assay developed within the WRAIR Malaria Vaccine Branch as described in Alemayehu et al. (2013). The PCR-based method will require only a minute amount of whole blood from a subject.

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

REFERENCES

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Sequences

The sequences below represent P. vivax and P. falciparum sequences, some of which including the indicated primers have been used in the examples described above. Modifications to the sequences are indicated.

GENBANK: XM_001608410.1 Plasmodium vivax SaI-1 ookinete surface protein Pvs25 partial mRNA; Corrected sequence from the original (altered nucleic have a double underline; underlined sequence is the restriction site) (SEQ ID NO: A2)

Original sequence of Pvs25: (SEQ ID NO: A3) ATGAACTCCTACTACAGCCTCTTCGTTTTTTTCCTCGTCCAAATTGCGCTAAAGTATAGCAAGGCAGC CGTCACGGTAGACACCATATGCAAAAATGGACAGCTGGTTCAAATGAGTAACCACTTTAAGTGTATGT GTAACGAAGGGCTGGTGCACCTTTCCGAAAATACATGTGAAGAAAAAAATGAATGCAAGAAAGAAACC CTAGGCAAAGCATGCGGGGAATTTGGCCAGTGTATAGAAAACCCAGACCCAGCACAGGTAAACATGTA CAAATGTGGTTGCATTGAGGGCTACACTTTGAAGGAAGACACTTGTGTGCTTGATGTATGTCAATACA AAAATTGTGGAGAAAGTGGCGAATGCATTGTTGAGTACCTCTCGGAAATCCAAAGTGCAGGTTGCTCA TGTGCTATTGGCAAAGTCCCCAATCCAGAAGATGAGAAAAAATGTACCAAAACGGGAGAAACTGCTTG TCAATTGAAATGTAACACAGATAATGAAGTCTGCAAAAATGTTGAAGGAGTTTACAAGTGCCAGTGTA TGGAAGGCTTTACGTTCGACAAAGAGAAAAATGTATGCCTTTCCTATTCTGTATTTAACATCCTAAAC TACTCCCTCTTCTTTATCATCCTGCTTGTCCTTTCGTACGTCATATAA Primers Used 5′ SacI E/L pro Pvs25 (SEQ ID NO: A4)

3′ XmaI E/L pro Pvs25 (SEQ ID NO: A6) GCGCATCCCGGGTTATATGACGTACGAAAGGACAAGCAGG GENBANK XM_001608411.1 Plasmodium vivax SaI-1 sexual stage surface protein Pvs28, partial mRNA (SEQ ID NO: A7) ATGAATACCTACCACAGCTTGCTGTTCCTTCTGGCCATCGTGCTTACTGTTAAGCACACCTTCGCAAAGGTCACC GCGGAGACCCAATGCAAAAATGGCTATGTAGTCCAAATGAGCAATCATTTTGAATGCAAATGCAACGACGGGTTT GTTCTGGCAAATGAAAACACTTGCGAGGAAAAACGCGATTGCACAAATCCACAAAATGTAAATAAAAACTGTGGA GACTACGCTGTGTGTGCAAACACCAGAATGAATAATGAGGAAAGAGCATTACGATGCGGCTGCATATTAGGGTAC ACCGTAATGAATGAGGTGTGTACTCCATATAAATGTAACGGCGTTCTGTGTGGAAAGGGAAAGTGCATCTTAGAT CCCGCTAATG TGAACAGCACCATGTGCTCTTGTAATATAGGAAGCACATTGGATGAATCTAAAAAATGTGGAAAGCCAGGAAAAA CTGAATGCACGTTGAAGTGTAAGGCAAACGAAGAATGTAAAGAGACTCAGAATTATTACAAGTGCGTTGCGAAGG GAAGCGGCGGAGAAGGCAGCGGTGGAGAAGGCAGCGGTGGAGAAGGCAGCGGCGGAGAGGGCAGCGGCGGAGAGG GCAGCGGTGGAGACACAGGAGCAGCTTACAGTCTCATGAACGGATCTGCAGTAATCAGCATACTACTTGTATTCG CCTTCTTCATGATGTCATTAGTGTAG 5′ EcoRI E/L pro Pvs28 (SEQ ID NO: A9)

3′ AatII E/L pro Pvs28 (SEQ ID NO: A12) CCGTCAGACGTCCTACACTAATGACATCATGAAGAAGGCG B13/14 GENBANK XM_001615397.1 Plasmodium vivax SaI-1 apical merozoite antigen 1, partial mRNA (SEQ ID NO: A13) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATAAAATATACTAC ATAATCTTTTTAAGCGCCCAGTGCCTTGTGCACATTGGGAAGTGCGGGCGAAACCAGAAGCCGAGCAGGCTGACC CGTAGCGCCAACAACGTTCTACTGGAAAAGGGGCCTACCGTTGAGAGAAGCACACGAATGAGTAACCCCTGGAAA GCGTTCATGGAAAAATACGACATCGAAAGAACACACAGTTCTGGGGTTCGAGTGGATTTAGGGGAAGATGCAGAA GTGGAAAATGCAAAGTACAGAATTCCAGCTGGAAGATGTCCTGTTTTTGGAAAGGGTATCGTTATAGAGAATTCT GACGTTAGCTTCTTAAGACCTGTGGCTACAGGAGATCAGAAGCTGAAGGATGGAGGTTTCGCCTTCCCCAATGCG AATGACCATATCTCCCCAATGACATTAGCGAACCTTAAGGAAAGGTATAAAGACAATGTAGAGATGATGAAGTTA AACGATATAGCTTTGTGCAGAACCCACGCAGCTAGCTTTGTCATGGCAGGGGATCAAAATTCGTCCTACAGACAC CCAGCTGTATACGACGAAAAGGAAAAAACATGCCACATGTTGTATTTATCAGCGCAGGAAAATATGGGTCCGAGG TACTGCAGCCCAGATGCACAAAATAGAGATGCCGTGTTCTGCTTCAAGCCAGATAAAAATGAAAGCTTTGAAAAC CTGGTGTATTTGAGCAAAAATGTGCGTAATGATTGGGATAAAAAATGCCCCCGTAAAAATTTAGGAAACGCCAAG TTCGGATTATGGGTGGATGGGAACTGCGAAGAAATTCCATACGTTAAAGAAGTGGAGGCAGAGGATCTGCGCGAA TGCAACCGAATCGTTTTCGGAGCGAGTGCCTCAGATCAACCAACTCAGTATGAAGAAGAAATGACGGATTATCAA AAAATACAACAAGGGTTTAGACAAAACAACCGAGAGATGATTAAAAGTGCCTTTCTTCCAGTGGGTGCATTCAAC TCGGATAATTTCAAAAGTAAAGGAAGAGGATTTAACTGGGCAAATTTCGATTCTGTAAAAAAGAAGTGTTACATT TTTAATACCAAACCGACTTGCCTCATTAATGACAAAAATTTTATTGCAACAACGGCGTTATCTCACCCACAAGAA GTAGACCTGGAGTTCCCCTGCAGCATATATAAAGACGAAATTGAAAGAGAAATTAAGAAACAATCGAGGAACATG AATCTGTACAGTGTTGATGGGGAACGCATTGTCCTGCCGAGGATATTTATCTCCAACGATAAGGAGAGTATCAAA TGTCCCTGCGAACCTGAGCGCATTTCCAACAGTACCTGCAACTTTTACGTTTGTAACTGTGTAGAGAAAAGGGCG GAAATTAAGGAAAATAACCAAGTTGTTATAAAGGAAGAATTTAGGGATTATTACGAAAATGGGGAGGAAAAATCG AACAAGCAGATGCTACTAATCATTATCGGAATAACTGGTGGCGTGTGCGTCGTCGCGCTGGCCTCTATGGCCTAC TTCAGGAAGAAGGCTAACAATGATAAGTATGACAAGATGGACCAGGCAGAGGGGTACGGGAAGCCCACCACCAGG AAGGACGAGATGCTCGACCCCGAGGCCTCCTTCTGGGGCGAGGACAAGCGGGCCTCCCACACCACGCCCGTGCTG ATGGAGAAGCCGTACTACTGACCTGCAGGATGGCGC  5′ BamHI E/L pro ama1 AMA gene primer-forward) (SEQ ID NO: A14)

3′ SbfI E/L pro ama1 (SEQ ID NO: A17) GCGCATCCTGCAGGTCAGTAGTACGGCTTCTCCATCAGCACGGGCG  A26 locus GENBANK AF063137.1 Plasmodium vivax clone B sporozoite surface protein 2 (SSP2) gene, complete cds (SEQ ID NO: A18)

(14NOV17: Change 5′ Enzyme from BamhI to MFE1 and 3′ from xhoI to Sbf) 5′ MfeI E/L pro Trap (SEQ ID NO: A19)

3′ Sbf E/L pro Trap (SEQ ID NO: A22) GCGCATCCTGCAGGTTAATTCCATTCGTTGTCTTCGGG I4L locus P42, GENBANK KF612323.1, Plasmodium vivax merozoite surface protein  1 (MSP1) gene, partial cds Bam pst/ecori and sbf are zero cutters (SEQ ID NO: A23) ATGGACCAAGTAACAACGGGAGAGGCAGAATCTGAGGCGCCTGAGATCCTCGTGCCAGCAGGAATCAGCGATTAC GATGTGGTCTACTTAAAGCCATTAGCCGGAATGTACAAAACGATAAAGAAGCAATTGGAAAATCACGTAAACGCA TTTAACACTAACATAACGGATATGTTAGACTCTAGACTGAAGAAGAGAAACTACTTCTTAGAAGTTCTGAACTCT GATTTGAACCCATTTAAGTATTCATCATCTGGTGAGTACATCATTAAGGACCCATACAAGCTGCTCGACTTGGAG AAGAAGAAGAAGCTTATAGGCAGCTACAAGTACATCGGTGCATCGATCGACATGGATCTGGCCACCGCGAATGAT GGCGTGACCTACTACAACAAGATGGGGGAGCTCTACAAGACGCACTTGGATGGAGTGAAAACAGAGATTAAGAAA GTCGAAGCTGATATTAAAGCAGAAGATGATAAGATTAAAACGATAGGAAGTGATAGCACTAAAACTACTGAAAAG ACCCAATCGATGGCCAAAAAGGCCGAGCTGGAGAAGTACCTCCCGTTCCTGAATAGCCTCCAAAAGGAGTACGAG TCCCTCGTGAGCAAGGTGAACACCTACACAGACAACCTAAAAAAAGTCATCAACAACTGCCAGCTGGAGAAAAAG GAAGCCGAGATCACTGTAAAGAAATTGCAGGACTACAACAAGATGGATGAGAAGTTGGAGGAGTACAAAAAATCG GAGAAAAAAAATGAAGTGAAGTCTTCTGGTCTTCTGGAAAAATTGATGAAATCAAAATTGATTAAAGAAAACGAG TCCAAGGAAATATTATCCCAGCTGCTAAATGTGCAAACTCAGTTATTAACTATGAGCTCCGAGCACACATGTATA GACACCAATGTGCCTGATAATGCAGCCTGCTATAGGTACTTGGACGGAACGGAAGAATGGAGATGCTTGTTAACC TTTAAAGAAGAAGGCGGCAAGTGTGTGCCAGCATCGAATGTGACTTGTAAGGATAACAATGGTGGTTGTGCCCCT GAAGCTGAATGTAAAATGACGGACAGCAATAAAATCGTCTGTAAATGTACTAAAGAAGGTTCTGAGCCACTCTTT GAGGGAGTTTTCTGTAGCTAA  5′ BamHI E/L pro msp1-42 (SEQ ID NO: A24)

3′ PstI E/L pro msp1-42 (SEQ ID NO: A26) GCGCATCTGCAGTTAGCTACAGAAAACTCCCTCAAAGAGTGGC Standard synthesis of vk210 original, with cloning into pUC57-Amp.  Standard synthesis of vk247 original, with cloning into pUC57-Amp. pUC57 vector sequence has recently been changed. vk210 original sequence: (SEQ ID NO: A27) AGTCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGCACAATGTAGATCTGTC CAAGGCCATAAATTTAAATGGAGTAAACTTCAATAATGTAGACGCCAGTTCACTTGGCGCGGCACACGTAGGACA AAGTGCTAGCCGAGGCAGAGGACTTGGTGAGAACCCAGATGACGAGGAAGGAGATGCTAAAAAAAAAAAGGATGG AAAGAAAGCAGAACCAAAAAATCCACGTGAAAATAAGCTGAAACAACCAGGAGACAGAGCAGATGGACAGCCAGC AGGAGACAGAGCAGATGGACAGCCAGCAGGTGATAGAGCAGATGGACAACCAGCAGGAGATAGAGCAGCTGGACA ACCAGCAGGAGATAGAGCAGATGGACAGCCAGCAGGAGACAGAGCAGATGGACAGCCAGCAGGAGACAGAGCAGA TGGACAACCAGCAGGAGACAGAGCAGATGGACAACCAGCAGGTGATAGAGCAGCTGGACAACCAGCAGGTGATAG AGCAGCTGGACAACCAGCAGGAGATAGAGCAGATGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGG AGATAGAGCAGATGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGATGGACAGCC AGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAGATAGAGCAGCTGG ACAGCCAGCAGGAGATAGAGCAGCTGGACAGCCAGCAGGAAATGGTGCAGGTGGACAGGCAGCAGGAGGAAACGC AGGAGGAGGACAGGGACAAAATAATGAAGGTGCGAATGCCCCAAATGAAAAGTCTGTGAAAGAATACCTAGATAA AGTTAGAGCTACCGTTGGCACCGAATGGACTCCATGCAGTGTAACCTGTGGAGTGGGTGTAAGAGTCAGAAGAAG AGTTAATGCAGCTAACAAAAAACCAGAGGATCTTACTTTGAATGACCTTGAGACTGATGTTTGTACAATGGATTA ACTGCAGAGTCAT  vk247 original: Length: 1114 (SEQ ID NO: A28) AGTCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGCACAATGTAGATCTGTC CAAGGCCATAAATTTAAATGGAGTAGGCTTCAATAATGTAGACGCCAGTTCACTTGGCGCGGCACACGTAGGACA AAGTGCTAGCCGAGGCAGAGGACTTGGTGAGAACCCAGATGACGAGGAAGGAGATGCTAAAAAAAAAAAGGATGG AAAGAAAGCAGAACCAAAAAATCCACGTGAAAATAAGCTGAAACAACCAGAAGATGGGGCAGGCAATCAACCAGG AGCAAATGGGGCTGGCAATCAACCAGGAGCAAATGGGGCAGGTAATCAACCAGGAGCAAATGGAGCAGGTGATCA ACCAGGAGCAAATGGGGCTGGCAATCAACCAGGAGCAAATGGAGCAGGTGATCAACCAGGAGCAAATGGGGCTGG CAATCAACCAGGAGCAAATGGGGCTGGCAATCAACCAGGAGCAAATGGGGCTGGCAATCAACCAGGAGCAAATGG AGCAGATGATCAACCAGGAGCAAATGGGGCAGGCAATCAACCAGGAGCAAATGGGGCTGGCAATCAACCAGGAGC AAATGGGGCAGGTAATCAACCAGGAGCAAATGGAGCAGGTGATCAACCAGGAGCAAATGGGGCTGGCAATCAACC AGGAGCAAATGGAGCAGGTGATCAACCAGGAGCAAATGGGGCCGGCAATCAACCAGGAGCAAATGGGGCAGGTAA TCAACCAGGAGCAAATGGGGCTGGCAATCAACCAGGAGCAAATGGGGCTGGTAATCAACCAGGAGCAAATGGAGC AGGTGGACAGGCAGCAGGAGGAAATGCTGCAAACAAAAAGGCAGGAGACGCAGGAGCAGGACAGGGACAAAATAA TGAAGGTGCGAATGCCACAAATGAAAAGTCTGTGAAAGAATACCTAGATAAAGTTAGAGCTACCGTTGGCACCGA ATGGACTCCATGCAGTGTAACCTGTGGAGTGGGTGTAAGAGTGAGAAGAAGAGTTAATGCAGCTAACAAAAAACC AGAGGATCTTACTTTGAATGACCTTGAGACTGATGTTTGTACAATGGATTAACTGCAGAGTCAT P.vivax-TRAP, with sequence verification and standard cloning into pUC57-Amp; P.vivax-TRAP: Length: 1742 (SEQ ID NO: A29) ATGCGCATCAATTGAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAGCTACTTCAGAAC AAAAGCTACCTGTTGGTGGTTTTCCTCCTGTACGTGAGTATATTCGCCAGAGGCGACGAAAAGGTTGTGGACGAA GTGAAGTATAGTGAGGAAGTATGTAACGAGAGCGTCGATTTATACCTCCTAGTTGACGGGTCTGGAAGTATTGGG TACCCGAATTGGATCACGAAGGTTATACCAATGCTCAACGGATTGATTAACAGCCTTAGCCTCTCAAGGGACACC ATCAACTTGTATATGAATTTATTTGGAAACTACACAACTGAATTGATAAGGCTTGGCAGTGGCCAGTCTATAGAC AAAAGACAAGCACTAAGTAAAGTTACAGAATTGAGAAAGACATACACACCTTACGGTACCACCAATATGACCGCT GCTCTGGATGAAGTGCAGAAGCATTTAAACGACCGAGTTAACAGAGAAAAAGCAATCCAACTGGTTATCCTAATG ACTGATGGTGTACCCAACAGTAAATACAGAGCCTTGGAGGTAGCCAATAAATTAAAGCAAAGAAATGTGAGTTTG GCTGTTATAGGAATTGGACAAGGCATAAACCACCAGTTCAATAGGCTAATAGCTGGGTGCCGCCCTCGTGAGCCA AACTGTAAATTTTATTCCTATGCTGACTGGAATGAGGCCGTAGCTCTCATCAAACCTTTTATTGCAAAGGTATGT ACTGAAGTGGAAAGAGTTGCTAACTGTGGTCCCTGGGACCCATGGACTGCGTGCAGCGTTACTTGTGGAAGGGGA ACCCACAGCAGGTCAAGACCATCGTTGCATGAAAAATGTACCACTCACATGGTTAGCGAATGTGAAGAGGGAGAA TGCCCTGTGGAACCTGAGCCTCTGCCCGTACCTGCTCCTCTCCCAACTGTCCCAGAAGATGTCAACCCACGTGAT ACGGATGATGAGAATGAAAACCCCAATTTTAACAAAGGCCTAGATGTGCCTGATGAAGATGATGATGAAGTTCCA CCTGCAAATGAAAGGGCGGATGGAAACCCAGTCGAAGAAAATGTCTTCCCACCAGCCGATGACTCTGTACCTGAT GAGTCAAACGTCCTTCCATTACCCCCTGCAGTTCCTGGAGGTTCCTCTGAGGAATTCCCAGCAGATGTTCAGAAT AACCCAGATAGCCCAGAAGAGTTACCAATGGAGCAGGAGGTGCCCCAAGACAACAACGTAAATGAACCAGAACGC TCAGACTCAAAAGGATATGGAGTGAACGAAAAGGTCATTCCAAACCCACTTGATAACGAAAGAGACATGGCCAAC AAGAACAAAACGGTTCATCCTGGTAGAAAAGACTCCGCTCGTGATAGGTACGCCAGACCACACGGAAGCACACAC GTAAACAATAACAGAGCAAACGAAAATTCGGACATTCCCAATAATCCCGTCCCATCTGACTACGAACAGCCTGAG GATAAAGCGAAGAAGTCATCAAATAATGGCTACAAAATTGCAGGTGGAGTGATTGCCGGGTTGGCTCTGGTCGGC TGCGTTGGATTCGCGTACAATTTCGTAGCTGGCGGAGGCGCCGCAGGAATGGCCGGTGAGCCTGCGCCCTTCGAT GAGGCGATGGCTGAGGATGAAAAAGACGTTGCGGAGGCGGACCAGTTCAAGCTGCCCGAAGACAACGAATGGAAT TAACCTGCAGGATGCGC  I4L P. vivax MSP1-42, I4L P.V-MSP1-42: Length: 2168 (SEQ ID NO: A30) ATGCTCACCGCGGTGTGGATTTCATTACTCATATTAATAATAAGCGTCTAAACACAATCTTGGTAATAGCAAAAG ATAACTTTTTAAAAAATTCTACATTTTCTGGAACTTTTATCAAAGAGAACATTATCTGGAAGGGTATCTATACTT ATAGAATAATCAAGTCTAGCTTTCCAGTTCCTACTATTAAGTCAGTTACTAATAAGAAAAAAATATGTAAGAAAC ATTGTTTCGTCAATTCTCAGTATACAACTAGGACTTTGTCACATATTCTTTGATCTAATTTTTAGATATAAATGG TGGATGCTATAACCGTTCTAACTGCGATCGGCATAACTGTATTAATGCTTTTGATGGTAATTTCTGGCGCCGCCA TGATAGTCAAGGAGTTAAATCCTAATGATATATTCACTATGCAATCATTAAAGTTTAATCGAGCCGTAACGATTT TCAAATATATAGGACTCTTTATCTATATACCAGGAACGATAATTTTGTACGCTACATACGTCAAATCCCTATTAA TGAAAAGTTAAATAATTTTTTTATTACACCAACAAAAGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAA TATAAATAAAAATGGACCAAGTAACAACGGGAGAGGCAGAATCTGAGGCGCCTGAGATCCTCGTGCCAGCAGGAA TCAGCGATTACGATGTGGTCTACTTAAAGCCATTAGCCGGAATGTACAAAACGATAAAGAAGCAATTGGAAAATC ACGTAAACGCATTTAACACTAACATAACGGATATGTTAGACTCTAGACTGAAGAAGAGAAACTACTTCTTAGAAG TTCTGAACTCTGATTTGAACCCATTTAAGTATTCATCATCTGGTGAGTACATCATTAAGGACCCATACAAGCTGC TCGACTTGGAGAAGAAGAAGAAGCTTATAGGCAGCTACAAGTACATCGGTGCATCGATCGACATGGATCTGGCCA CCGCGAATGATGGCGTGACCTACTACAACAAGATGGGGGAGCTCTACAAGACGCACTTGGATGGAGTGAAAACAG AGATTAAGAAAGTCGAAGCTGATATTAAAGCAGAAGATGATAAGATTAAAACGATAGGAAGTGATAGCACTAAAA CTACTGAAAAGACCCAATCGATGGCCAAAAAGGCCGAGCTGGAGAAGTACCTCCCGTTCCTGAATAGCCTCCAAA AGGAGTACGAGTCCCTCGTGAGCAAGGTGAACACCTACACAGACAACCTAAAAAAAGTCATCAACAACTGCCAGC TGGAGAAAAAGGAAGCCGAGATCACTGTAAAGAAATTGCAGGACTACAACAAGATGGATGAGAAGTTGGAGGAGT ACAAAAAATCGGAGAAAAAAAATGAAGTGAAGTCTTCTGGTCTTCTGGAAAAATTGATGAAATCAAAATTGATTA AAGAAAACGAGTCCAAGGAAATATTATCCCAGCTGCTAAATGTGCAAACTCAGTTATTAACTATGAGCTCCGAGC ACACATGTATAGACACCAATGTGCCTGATAATGCAGCCTGCTATAGGTACTTGGACGGAACGGAAGAATGGAGAT GCTTGTTAACCTTTAAAGAAGAAGGCGGCAAGTGTGTGCCAGCATCGAATGTGACTTGTAAGGATAACAATGGTG GTTGTGCCCCTGAAGCTGAATGTAAAATGACGGACAGCAATAAAATCGTCTGTAAATGTACTAAAGAAGGTTCTG AGCCACTCTTTGAGGGAGTTTTCTGTAGCTAACTGCAGACAAAAACATTTTTATTCTCAAATGAGATAAAGTGAA AATATATATCATTATATTACAAAGTACAATTATTTAGGTTTAATCATGAGTAAGGTAATCAAGAAGAGAGTTGAA ACTTCACCAAGACCTACTGCATCTAGCGATTCTCTACAGACTTGTGCGGGTGTTATAGAGTATGCAAAATCGATT AGTAAATCTAATGCAAAATGTATCGAATACGTTACACTAAATGCTTCTCAATACGCTAATTGTTCGTCTATCTCT ATAAAACTTACTGATAGTTTATCTAGTCAAATGACTTCCACTTTTATTATGTTGGAAGGAGAGACTAAACTTTAT AAAAATAAATCTAAACAAGATAGAAGCGATGGATACTTTCTAAAAATAAAAGTTACCCGGGATGCTCA P.vivax AMA1 gene (SEQ ID NO: A31) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATAAAATATACTAC ATAATCTTTTTAAGCGCCCAGTGCCTTGTGCACATTGGGAAGTGCGGGCGAAACCAGAAGCCGAGCAGGCTGACC CGTAGCGCCAACAACGTTCTACTGGAAAAGGGGCCTACCGTTGAGAGAAGCACACGAATGAGTAACCCCTGGAAA GCGTTCATGGAAAAATACGACATCGAAAGAACACACAGTTCTGGGGTTCGAGTGGATTTAGGGGAAGATGCAGAA GTGGAAAATGCAAAGTACAGAATTCCAGCTGGAAGATGTCCTGTTTTTGGAAAGGGTATCGTTATAGAGAATTCT GACGTTAGCTTCTTAAGACCTGTGGCTACAGGAGATCAGAAGCTGAAGGATGGAGGTTTCGCCTTCCCCAATGCG AATGACCATATCTCCCCAATGACATTAGCGAACCTTAAGGAAAGGTATAAAGACAATGTAGAGATGATGAAGTTA AACGATATAGCTTTGTGCAGAACCCACGCAGCTAGCTTTGTCATGGCAGGGGATCAAAATTCGTCCTACAGACAC CCAGCTGTATACGACGAAAAGGAAAAAACATGCCACATGTTGTATTTATCAGCGCAGGAAAATATGGGTCCGAGG TACTGCAGCCCAGATGCACAAAATAGAGATGCCGTGTTCTGCTTCAAGCCAGATAAAAATGAAAGCTTTGAAAAC CTGGTGTATTTGAGCAAAAATGTGCGTAATGATTGGGATAAAAAATGCCCCCGTAAAAATTTAGGAAACGCCAAG TTCGGATTATGGGTGGATGGGAACTGCGAAGAAATTCCATACGTTAAAGAAGTGGAGGCAGAGGATCTGCGCGAA TGCAACCGAATCGTTTTCGGAGCGAGTGCCTCAGATCAACCAACTCAGTATGAAGAAGAAATGACGGATTATCAA AAAATACAACAAGGGTTTAGACAAAACAACCGAGAGATGATTAAAAGTGCCTTTCTTCCAGTGGGTGCATTCAAC TCGGATAATTTCAAAAGTAAAGGAAGAGGATTTAACTGGGCAAATTTCGATTCTGTAAAAAAGAAGTGTTACATT TTTAATACCAAACCGACTTGCCTCATTAATGACAAAAATTTTATTGCAACAACGGCGTTATCTCACCCACAAGAA GTAGACCTGGAGTTCCCCTGCAGCATATATAAAGACGAAATTGAAAGAGAAATTAAGAAACAATCGAGGAACATG AATCTGTACAGTGTTGATGGGGAACGCATTGTCCTGCCGAGGATATTTATCTCCAACGATAAGGAGAGTATCAAA TGTCCCTGCGAACCTGAGCGCATTTCCAACAGTACCTGCAACTTTTACGTTTGTAACTGTGTAGAGAAAAGGGCG GAAATTAAGGAAAATAACCAAGTTGTTATAAAGGAAGAATTTAGGGATTATTACGAAAATGGGGAGGAAAAATCG AACAAGCAGATGCTACTAATCATTATCGGAATAACTGGTGGCGTGTGCGTCGTCGCGCTGGCCTCTATGGCCTAC TTCAGGAAGAAGGCTAACAATGATAAGTATGACAAGATGGACCAGGCAGAGGGGTACGGGAAGCCCACCACCAGG AAGGACGAGATGCTCGACCCCGAGGCCTCCTTCTGGGGCGAGGACAAGCGGGCCTCCCACACCACGCCCGTGCTG ATGGAGAAGCCGTACTACTGACCTGCAGGATGCGC C7K1L-P. vivax DBP, RII-PVS28-PVS25 (3 genes): Length: 3536 bp (SEQ ID NO: A32) AGCTACCCGCGGGTTTGCATCGTGCTTTAACATCAATGGTACAAATTTTATCCTCGCTTTGTGTATCATATTCGT CCCTACTATAAAATTGTATATTCAGATTATCATGAGATGTGTATACGCTAACGGTATCAATAAACGGAGCACACC ATTTAGTCATAACCGTAATCCAAAAATTTTTAAAGTATATCTTAACGAAAGAAGTTGTATCATCGTTAGGATTTG GTAAATCATTATCTACAGTGTATGGTACTAGATCCTCATAAGTGTATATATCTAGAGTAATGTTTAATTTATCAA ATGGTTGATAATATGGATCCTCATGACAATTTCCGAAGATGGAAATGAGATATAGACATGCAATAAATCTAATCG AAGACATGGTTACTCCTTAAAAAAATACGAATAATCACCTTGGCTATTTAGTAAGTGTCATTTAACACTATACTC ATAGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATCATGCTTTCCTCCAAAA TACTGTAATGAAAAACTGTAATTATAAGAGAAAACGTCGGGAAAGAGATTGGGACTGTAACACTAAGAAGGATGT TTGTATACCAGATCGAAGATATCAATTATGTATGAAGGAACTTACGAATTTGGTAAATAATACAGACACAAATTT TCATAGGGATATAACATTTCGAAAATTATATTTGAAAAGGAAACTTATTTATGATGCTGCAGTAGAGGGCGATTT ATTACTTAAGTTGAATAACTACAGATATAACAAAGACTTTTGCAAGGATATAAGATGGAGTTTGGGAGATTTTGG AGATATAATTATGGGAACGGATATGGAAGGCATCGGATATTCCAAAGTAGTGGAAAATAATTTGCGCAGCATCTT TGGAACTGATGAAAAGGCCCAACAGCGTCGTAAACAGTGGTGGAATGAATCTAAAGCACAAATTTGGACAGCAAT GATGTACTCAGTTAAAAAAAGATTAAAGGGGAATTTTATATGGATTTGTAAATTAAATGTTGCGGTAAATATAGA ACCGCAGATATATAGATGGATTCGAGAATGGGGAAGGGATTACGTGTCAGAATTGCCCACAGAAGTGCAAAAACT GAAAGAAAAATGTGATGGAAAAATCAATTATACTGATAAAAAAGTATGTAAGGTACCACCATGTCAAAATGCGTG TAAATCATATGATCAATGGATAACCAGAAAAAAAAATCAATGGGATGTTCTGTCAAATAAATTCATAAGTGTAAA AAACGCAGAAAAGGTTCAGACGGCAGGTATCGTAACTCCTTATGATATACTAAAACAGGAGTTAGATGAATTTAA CGAGGTGGCTTTTGAGAATGAAATTAACAAACGTGATGGTGCATATATTGAGTTATGAGACGTCCCTGCAGGAGC TCGGATCGAATCATAAAAAAATATATTATTTTTATGTTATTTTTGTAGCGCTAAAAATTGAAATTTTATTTTTTT TTTTTGGAATATAAATAAAAATGAATACCTACCACAGCTTGCTGTTCCTTCTGGCCATCGTGCTTACTGTTAAGC ACACCTTCGCAAAGGTCACCGCTGAAACCCAATGCAAAAATGGCTATGTAGTCCAAATGAGCAATCATTTTGAAT GCAAATGCAACGACGGGTTTGTTCTGGCAAATGAAAACACTTGCGAGGAAAAACGCGATTGCACAAATCCACAAA ATGTAAATAAAAACTGTGGAGACTACGCTGTGTGTGCAAACACCAGAATGAATAATGAGGAAAGAGCATTACGAT GCGGCTGCATATTAGGGTACACCGTAATGAATGAGGTGTGTACTCCATATAAATGTAACGGCGTTCTGTGTGGAA AGGGAAAGTGCATCTTAGATCCCGCTAATGTGAACAGCACCATGTGCTCTTGTAATATAGGAAGCACATTGGATG AATCTAAAAAATGTGGAAAGCCAGGAAAAACTGAATGCACGTTGAAGTGTAAGGCAAACGAAGAATGTAAAGAGA CTCAGAATTATTACAAGTGCGTTGCGAAGGGAAGCGGCGGAGAAGGCAGCGGTGGAGAAGGCAGCGGTGGAGAAG GCAGCGGCGGAGAGGGCAGCGGCGGAGAGGGCAGCGGTGGAGACACAGGAGCAGCTTACAGTCTCATGAACGGAT CTGCAGTAATCAGCATACTACTTGTATTCGCCTTCTTCATGATGTCATTAGTGTAGGGCGCCTATTGGTTAAAAA TGAAAATGGATTTTTATTTTATGCGGTTATCTTTTTGTACCGTACAATCGCGCGCAAAAATTGAAATTTTATTTT TTTTTTTTGGAATATAAATAAAAATGAACTCCTACTACAGCCTCTTCGTCTTCTTCCTCGTCCAAATTGCGCTAA AGTATAGCAAGGCAGCCGTCACGGTAGACACCATATGCAAAAATGGACAGCTGGTTCAAATGAGTAACCACTTTA AGTGTATGTGTAACGAAGGGCTGGTGCACCTTTCCGAAAATACATGTGAAGAAAAAAATGAATGCAAGAAAGAAA CCCTAGGCAAAGCATGCGGGGAATTTGGCCAGTGTATAGAAAACCCAGACCCAGCACAGGTAAACATGTACAAAT GTGGTTGCATTGAGGGCTACACTTTGAAGGAAGACACTTGTGTGCTTGATGTATGTCAATACAAAAATTGTGGAG AAAGTGGCGAATGCATTGTTGAGTACCTCTCGGAAATCCAAAGTGCAGGTTGCTCATGTGCTATTGGCAAAGTCC CCAATCCAGAAGATGAGAAAAAATGTACCAAAACGGGAGAAACTGCTTGTCAATTGAAATGTAACACAGATAATG AAGTCTGCAAAAATGTTGAAGGAGTTTACAAGTGCCAGTGTATGGAAGGCTTTACGTTCGACAAAGAGAAAAATG TATGCCTTTCCTATTCTGTATTTAACATCCTAAACTACTCCCTCTTCTTTATCATCCTGCTTGTCCTTTCGTACG TCATATAACCTGCAGGAAAAATAATATTTATTAAGAAAATTCAGATTTCACGTACCCATCAATATAAATAAAATA ATGATTCCTTCCACCGTATCCATAAACAATATTAAGGAGATTCTACCTTACCCATAAACAATATAAATCCAGTAA TATCATGTCTAATGATGAACACAAATGGTGTATTAAATTCCAGTTTTTCAGGAGATGATCTCGCCGTAGCTACCA TGATAGTAGATGCCTCTGCTACAGTTCCTTGTTCGTCGACATCTATCTTTGCATTCTGAAACATTTTATAAATAT ATAATGGGTCCCTAGTCATATGTTTAAACGACGCATTATCTGGATTAAACATACTAGGAGCCATCATTTCGGCTA TCGACTTAATATCCCTCTTATTTTCGATAGAAAATTTAGGGAGTTTAAGATTGTACACTTTATTCCCTAATTGAA ACGACCAATAGTCTAATTTTGCAGCCGTAATAGAATCTGTGAAATGGGTCATATTATCACCTATTGCCAGGTACG CCGGCAGCTAC  C7K1L P. vivax DBPRII-PVS28-PVS25 (3536 BP, 3 genes) LEFT ARM SacII AND RIGHT ARM NgoMIV (SEQ ID NO: B1.2) AGCTACCCGCGGGtttgcatcgtgctttaacatcaatggtacaaattttatcctcgctttgtgtatca tattcgtcctAtggttactccttaaaaaaatacgaataatcaccttggctatttagtaagtgTcattt aacactatactcatagaattcAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGA ATCATGCTTTCCTCCAAAATACTGTAATGAAAAACTGTAATTATAAGAGAAAACGTCGGGAAAGAGAT TGGGACTGTAACACTAAGAAGGATGTTTGTATACCAGATCGAAGATATCAATTATGTATGAAGGAACT TACGAATTTGGTAAATAATACAGACACAAATTTTCATAGGGATATAACATTTCGAAAATTATATTTGA AAAGGAAACTTATTTATGATGCTGCAGTAGAGGGCGATTTATTACTTAAGTTGAATAACTACAGATAT AACAAAGACTTTTGCAAGGATATAAGATGGAGTTTGGGAGATTTTGGAGATATAATTATGGGAACGGA TATGGAAGGCATCGGATATTCCAAAGTAGTGGAAAATAATTTGCGCAGCATCTTTGGAACTGATGAAA AGGCCCAACAGCGTCGTAAACAGTGGTGGAATGAATCTAAAGCACAAATTTGGACAGCAATGATGTAC TCAGTTAAAAAAAGATTAAAGGGGAATTTTATATGGATTTGTAAATTAAATGTTGCGGTAAATATAGA ACCGCAGATATATAGATGGATTCGAGAATGGGGAAGGGATTACGTGTCAGAATTGCCCACAGAAGTGC AAAAACTGAAAGAAAAATGTGATGGAAAAATCAATTATACTGATAAAAAAGTATGTAAGGTACCACCA TGTCAAAATGCGTGTAAATCATATGATCAATGGATAACCAGAAAAAAAAATCAATGGGATGTTCTGTC AAATAAATTCATAAGTGTAAAAAACGCAGAAAAGGTTCAGACGGCAGGTATCGTAACTCCTTATGATA TACTAAAACAGGAGTTAGATGAATTTAACGAGGTGGCTTTTGAGAATGAAATTAACAAACGTGATGGT GCATATATTGAGTTATGAGacgtcagcgctAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAA TAAAAATGAATACCTACCACAGCTTGCTGTTCCTTCTGGCCATCGTGCTTACTGTTAAGCACACCTTC GCAAAGGTCACCGCTGAAACCCAATGCAAAAATGGCTATGTAGTCCAAATGAGCAATCATTTTGAATG CAAATGCAACGACGGGTTTGTTCTGGCAAATGAAAACACTTGCGAGGAAAAACGCGATTGCACAAATC CACAAAATGTAAATAAAAACTGTGGAGACTACGCTGTGTGTGCAAACACCAGAATGAATAATGAGGAA AGAGCATTACGATGCGGCTGCATATTAGGGTACACCGTAATGAATGAGGTGTGTACTCCATATAAATG TAACGGCGTTCTGTGTGGAAAGGGAAAGTGCATCTTAGATCCCGCTAATGTGAACAGCACCATGTGCT CTTGTAATATAGGAAGCACATTGGATGAATCTAAAAAATGTGGAAAGCCAGGAAAAACTGAATGCACG TTGAAGTGTAAGGCAAACGAAGAATGTAAAGAGACTCAGAATTATTACAAGTGCGTTGCGAAGGGAAG CGGCGGAGAAGGCAGCGGTGGAGAAGGCAGCGGTGGAGAAGGCAGCGGCGGAGAGGGCAGCGGCGGAG AGGGCAGCGGTGGAGACACAGGAGCAGCTTACAGTCTCATGAACGGATCTGCAGTAATCAGCATACTA CTTGTATTCGCCTTCTTCATGATGTCATTAGTGTAGggcgccGCGCGCAAAAATTGAAATTTTATTTT TTTTTTTTGGAATATAAATAAAAATGAACTCCTACTACAGCCTCTTCGTCTTCTTCCTCGTCCAAATT GCGCTAAAGTATAGCAAGGCAGCCGTCACGGTAGACACCATATGCAAAAATGGACAGCTGGTTCAAAT GAGTAACCACTTTAAGTGTATGTGTAACGAAGGGCTGGTGCACCTTTCCGAAAATACATGTGAAGAAA AAAATGAATGCAAGAAAGAAACCCTAGGCAAAGCATGCGGGGAATTTGGCCAGTGTATAGAAAACCCA GACCCAGCACAGGTAAACATGTACAAATGTGGTTGCATTGAGGGCTACACTTTGAAGGAAGACACTTG TGTGCTTGATGTATGTCAATACAAAAATTGTGGAGAAAGTGGCGAATGCATTGTTGAGTACCTCTCGG AAATCCAAAGTGCAGGTTGCTCATGTGCTATTGGCAAAGTCCCCAATCCAGAAGATGAGAAAAAATGT ACCAAAACGGGAGAAACTGCTTGTCAATTGAAATGTAACACAGATAATGAAGTCTGCAAAAATGTTGA AGGAGTTTACAAGTGCCAGTGTATGGAAGGCTTTACGTTCGACAAAGAGAAAAATGTATGCCTTTCCT ATTCTGTATTTAACATCCTAAACTACTCCCTCTTCTTTATCATCCTGCTTGTCCTTTCGTACGTCATA TAAcctgcaggaaaaataatatttattaagaaaattcagatttcacgTacccatcaatataaataaaa taatgattccttccaccgtatccataaacaataGCCGGCagctac 5′WPP C7.K1 remove IGR SbfI (SEQ ID NO: B1.1) GATGGTGCATATATTGAGTTATGAGACGTC 3′WVPP C7.K1 remove IGR Sbf1 (SEQ ID NO: B1) CAAACGTGATGGTGCATATATTGAGTTATGAGACGTCGTTTGCACTACCACGTATATAACTCAATA CTCTGCAGTCGAGCCCCGAGCTGACGTCTCATAACTCAATATATGCACCATCACGTTTG I4L locus; GENBANK KF612323.1, Plasmodium vivax merozoite surface protein 1   (MSP1) gene, partial cds Bam pst/ecori and sbf are zero cutters; introduced restriction enzyme site is  underlined I4L P. vivax MSP1-42 (2168 BP) I4L left Arm sacII and right arm xma1 (SEQ ID NO: B2) ATGCTCACCGCGGTGTGGATTTCATTACTCATATTAATAATAAGCGTCTAAACACAATCTTGGTAATAGCAAAAG ATAACTTTTTAAAAAATTCTACATTTTCTGGAACTTTTATCAAAGAGAACATTATCTGGAAGGGTATCTATACTT ATAGAATAATCAAGTCTAGCTTTCCAGTTCCTACTATTAAGTCAGTTACTAATAAGAAAAAAATATGTAAGAAAC ATTGTTTCGTCAATTCTCAGTATACAACTAGGACTTTGTCACATATTCTTTGATCTAATTTTTAGATATAAATGG TGGATGCTATAACCGTTCTAACTGCGATCGGCATAACTGTATTAATGCTTTTGATGGTAATTTCTGGCGCCGCCA TGATAGTCAAGGAGTTAAATCCTAATGATATATTCACTATGCAATCATTAAAGTTTAATCGAGCCGTAACGATTT TCAAATATATAGGACTCTTTATCTATATACCAGGAACGATAATTTTGTACGCTACATACGTCAAATCCCTATTAA TGAAAAGTTAAATAATTTTTTTATTACACCAACAAAAGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAA TATAAATAAAAATGGACCAAGTAACAACGGGAGAGGCAGAATCTGAGGCGCCTGAGATCCTCGTGCCAGGAGGAA TCAGCGATTACGATGTGGTCTACTTAAAGCCATTAGCCGGAATGTACAAAACGATAAAGAAGCAATTGGAAAATC ACGTAAACGCATTTAACACTAACATAACGGATATGTTAGACTCTAGACTGAAGAAGAGAAACTACTTCTTAGAAG TTCTGAACTCTGATTTGAACCCATTTAAGTATTCATCATCTGGTGAGTACATCATTAAGGACCCATACAAGCTGC TCGACTTGGAGAAGAAGAAGAAGCTTATAGGCAGCTACAAGTACATCGGTGCATCGATCGACATGGATCTGGCCA CCGCGAATGATGGCGTGACCTACTACAACAAGATGGGGGAGCTCTACAAGACGCACTTGGATGGAGTGAAAACAG AGATTAAGAAAGTCGAAGCTGATATTAAAGCAGAAGATGATAAGATTAAAACGATAGGAAGTGATAGCACTAAAA CTACTGAAAAGACCCAATCGATGGCCAAAAAGGCCGAGCTGGAGAAGTACCTCCCGTTCCTGAATAGCCTCCAAA AGGAGTACGAGTCCCTCGTGAGCAAGGTGAACACCTACACAGACAACCTAAAAAAAGTCATCAACAACTGCCAGC TGGAGAAAAAGGAAGCCGAGATCACTGTAAAGAAATTGCAGGACTACAACAAGATGGATGAGAAGTTGGAGGAGT ACAAAAAATCGGAGAAAAAAAATGAAGTGAAGTCTTCTGGTCTTCTGGAAAAATTGATGAAATCAAAATTGATTA AAGAAAACGAGTCCAAGGAAATATTATCCCAGCTGCTAAATGTGCAAACTCAGTTATTAACTATGAGCTCCGAGC ACACATGTATAGACACCAATGTGCCTGATAATGCAGCCTGCTATAGGTACTTGGACGGAACGGAAGAATGGAGAT GCTTGTTAACCTTTAAAGAAGAAGGCGGCAAGTGTGTGCCAGCATCGAATGTGACTTGTAAGGATAACAATGGTG GTTGTGCCCCTGAAGCTGAATGTAAAATGACGGACAGCAATAAAATCGTCTGTAAATGTACTAAAGAAGGTTCTG AGCCACTCTTTGAGGGAGTTTTCTGTAGCTAActgcagACAAAAACATTTTTATTCTCAAATGAGATAAAGTGAA AATATATATCATTATATTACAAAGTACAATTATTTAGGTTTAATCATGAGTAAGGTAATCAAGAAGAGAGTTGAA ACTTCACCAAGACCTACTGCATCTAGCGATTCTCTACAGACTTGTGCGGGTGTTATAGAGTATGCAAAATCGATT AGTAAATCTAATGCAAAATGTATCGAATACGTTACACTAAATGCTTCTCAATACGCTAATTGTTCGTCTATCTCT ATAAAACTTACTGATAGTTTATCTAGTCAAATGACTTCCACTTTTATTATGTTGGAAGGAGAGACTAAACTTTAT AAAAATAAATCTAAACAAGATAGAAGCGATGGATACTTTCTAAAAATAAAAGTTACCCGGGATGCTCA Pv28

Pv28 Protein (SEQ ID NO: C1) MNTYHSLLFLLAIVLTVKHTFAKVTAETQCKNGYVVQMSNHFECKCNDGFVLANENTCEEKRDCTNPQNVNKNCG DYAVCANTRMNNEERALRCGCILGYTVMNEVCTPYKCNGVLCGKGKCILDPANVNSTMCSCNIGSTLDESKKCGK PGKTECTLKCKANEECKETQNYYKCVAKGSGGEGSGGEGSGGEGSGGEGSGGEGSGGDTGAAYSLMNGSAVISIL LVFAFFMMSLV Pv-28 gene after changing 2 nucleotides to a silent mutation (changed nucleotides  are underlined) (SEQ ID NO: C2)

P. vivax CS-VK210 (SEQ ID NO: C4)

P. vivax CS-VK247 gene, native sequence (SEQ ID NO: D4)

(SEQ ID NO: D5)

P. vivax CS-VK247 protein (SEQ ID NO: D6) MHNVDLSKAINLNGVGFNNVDASSLGAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEP KNPRENKLKQPEDGAGNQPGANGAGNQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGD QPGANGAGNQPGANGAGNQPGANGAGNQPGANGADDQPGANGAGNQPGANGAGNQPGANGAG NQPGANGAGDQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA GNQPGANGAGGQAAGGNAANKKAGDAGAGQGQNNEGANATNEKSVKEYLDKVRATVGTEWTP CSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTMD  gi|124804477|ref|XM_001347979.1| Plasmodium falciparum 3D7 apical membrane antigen 1, AMA1 (AMA1) mRNA, complete cds (SEQ ID NO: F1) ATGAGAAAATTATACTGCGTATTATTATTGAGCGCCTTTGAGTTTACATATATGATAAACTTTGGAAGAGGACAG AATTATTGGGAACATCCATATCAAAATAGTGATGTGTATCGTCCAATCAACGAACATAGGGAACATCCAAAAGAA TACGAATATCCATTACACCAGGAACATACATACCAACAAGAAGATTCAGGAGAAGACGAAAATACATTACAACAC GCATATCCAATAGACCACGAAGGTGCCGAACCCGCACCACAAGAACAAAATTTATTTTCAAGCATTGAAATAGTA GAAAGAAGTAATTATATGGGTAATCCATGGACGGAATATATGGCAAAATATGATATTGAAGAAGTTCATGGTTCA GGTATAAGAGTAGATTTAGGAGAAGATGCTGAAGTAGCTGGAACTCAATATAGACTTCCATCAGGGAAATGTCCA GTATTTGGTAAAGGTATAATTATTGAGAATTCAAATACTACTTTTTTAACACCGGTAGCTACGGGAAATCAATAT TTAAAAGATGGAGGTTTTGCTTTTCCTCCAACAGAACCTCTTATGTCACCAATGACATTAGATGAAATGAGACAT TTTTATAAAGATAATAAATATGTAAAAAATTTAGATGAATTGACTTTATGTTCAAGACATGCAGGAAATATGATT CCAGATAATGATAAAAATTCAAATTATAAATATCCAGCTGTTTATGATGACAAAGATAAAAAGTGTCATATATTA TATATTGCAGCTCAAGAAAATAATGGTCCTAGATATTGTAATAAAGACGAAAGTAAAAGAAACAGCATGTTTTGT TTTAGACCAGCAAAAGATATATCATTTCAAAACTATACATATTTAAGTAAGAATGTAGTTGATAACTGGGAAAAA GTTTGCCCTAGAAAGAATTTACAGAATGCAAAATTCGGATTATGGGTCGATGGAAATTGTGAAGATATACCACAT GTAAATGAATTTCCAGCAATTGATCTTTTTGAATGTAATAAATTAGTTTTTGAATTGAGTGCTTCGGATCAACCT AAACAATATGAACAACATTTAACAGATTATGAAAAAATTAAAGAAGGTTTCAAAAATAAGAACGCTAGTATGATC AAAAGTGCTTTTCTTCCCACTGGTGCTTTTAAAGCAGATAGATATAAAAGTCATGGTAAGGGTTATAATTGGGGA AATTATAACACAGAAACACAAAAATGTGAAATTTTTAATGTCAAACCAACATGTTTAATTAACAATTCATCATAC ATTGCTACTACTGCTTTGTCCCATCCCATCGAAGTTGAAAACAATTTTCCATGTTCATTATATAAAGATGAAATA ATGAAAGAAATCGAAAGAGAATCAAAACGAATTAAATTAAATGATAATGATGATGAAGGGAATAAAAAAATTATA GCTCCAAGAATTTTTATTTCAGATGATAAAGACAGTTTAAAATGCCCATGTGACCCTGAAATGGTAAGTAATAGT ACATGTCGTTTCTTTGTATGTAAATGTGTAGAAAGAAGGGCAGAAGTAACATCAAATAATGAAGTTGTAGTTAAA GAAGAATATAAAGATGAATATGCAGATATTCCTGAACATAAACCAACTTATGATAAAATGAAAATTATAATTGCA TCATCAGCTGCTGTCGCTGTATTAGCAACTATTTTAATGGTTTATCTTTATAAAAGAAAAGGAAATGCTGAAAAA TATGATAAAATGGATGAACCACAAGATTATGGGAAATCAAATTCAAGAAATGATGAAATGTTAGATCCTGAGGCA TCTTTTTGGGGGGAAGAAAAAAGAGCATCACATACAACACCAGTTCTGATGGAAAAACCATACTATTAA 5′ BamHI Pf3D7 E/L pro AMA1 (SEQ ID NO: F2) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAGAAAATTATACTGC GTATTATTATTGAGC 3′ PstI Pf3D7 AMA1 (SEQ ID NO: F3) GCGCATCTGCAGTTAATAGTATGGTTTTTCCATCAG 5′ AMA1 WPP TTS seq 275 (SEQ ID NO: F4) GATGAAATGAGACACTTCTATAAAG 3′ AMA1 WPP TTS seq 275 (SEQ ID NO: F5) CATATTTATTATCTTTATAGAAGTGTCTCATTTC 5′ AMA1 WPP TTS seq 1435 (SEQ ID NO: F6) ATAGCTCCAAGAATCTTCATTTCAGATGATAAAGAC 3′ AMA1 WPP TTS seq 1435 (SEQ ID NO: F7) GGGCATTTTAAACTGTCTTTATCATCTGAAATGAAGATTCTTGGAGCTATAATTTTTTTATTCCC >gi|124805897|ref|XM_001350533.1| Plasmodium falciparum 3D7 CelTOS, putative (CelTOS) mRNA, complete cds (SEQ ID NO: F8) ATGAATGCCTTAAGAAGATTACCAGTTATTTGCTCTTTCTTAGTATTTCTTGTCTTTTCCAATGTTTT AT GTTTCAGAGGAAACAACGGACACAATTCTTCATCATCTCTCTATAATGGAAGCCAATTTATTGAACAA TT AAATAACAGTTTTACTTCAGCTTTTCTTGAATCACAATCAATGAATAAGATTGGTGATGATTTAGCAG AG ACCATATCAAATGAACTTGTCAGTGTTTTACAAAAAAATTCACCAACCTTTTTAGAATCAAGCTTTGA TA TCAAATCAGAAGTAAAAAAACACGCAAAATCTATGTTAAAGGAATTAATCAAAGTAGGATTGCCATCA TT CGAAAATCTCGTAGCTGAAAATGTTAAACCACCAAAAGTCGACCCAGCAACATATGGTATAATAGTAC CA GTATTAACATCTTTATTTAATAAGGTAGAAACAGCTGTAGGTGCGAAAGTTTCTGATGAGATATGGAA TT ACAATTCACCAGACGTCTCAGAAAGTGAAGAAAGTTTATCAGATGATTTTTTCGATTAA 5′ BamHI Pf3D7 E/L pro CelTOS (SEQ ID NO: F9) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATGCCTT AAGAAGATTACC 3′ XhoI Pf3D7 CelTOS (SEQ ID NO: F10) GCGCATCTCGAGTTAATCGAAAAAATCATCTG gi|124504758; XM_001351086.1; Plasmodium falciparum 3D7  circumsporozoite (CS) protein (PFC0210c) mRNA, complete cds (SEQ ID NO: F11) ATGATGAGAAAATTAGCTATTTTATCTGTTTCTTCCTTTTTATTTGTTGAGGCCTTATTCCAGGAATA CCAG GCTATGGAAGTTCGTCAAACACAAGGGTTCTAAATGAATTAAATTATGATAATGCAGGCACTAATTT ATATAATGAATTAGAAATGAATTATTATGGGAAACAGGAAAATTGGTATAGTCTTAAAAAAAATAGTA GA TCACTTGGAGAAAATGATGATGGAAATAACGAAGACAACGAGAAATTAAGGAAACCAAAACATAAAAA AT TAAAGCAACCAGCGGATGGTAATCCTGATCCAAATGCAAACCCAAATGTAGATCCCAATGCCAACCCA AA TGTAGATCCAAATGCAAACCCAAATGTAGATCCAAATGCAAACCCAAATGCAAACCCAAATGCAAACC CA AATGCAAACCCAAATGCAAACCCAAATGCAAACCCAAATGCAAACCCAAATGCAAACCCAAATGCAAA CC CAAATGCAAACCCAAATGCAAACCCAAATGCAAACCCAAATGCAAACCCAAATGCAAACCCCAATGCA AA TCCTAATGCAAACCCAAATGCAAACCCAAACGTAGATCCTAATGCAAATCCAAATGCAAACCCAAACG CA AACCCCAATGCAAATCCTAATGCAAACCCCAATGCAAATCCTAATGCAAATCCTAATGCCAATCCAAA TG CAAATCCAAATGCAAACCCAAACGCAAACCCCAATGCAAATCCTAATGCCAATCCAAATGCAAATCCA AA TGCAAACCCAAATGCAAACCCAAATGCAAACCCCAATGCAAATCCTAATAAAAACAATCAAGGTAATG GA CAAGGTCACAATATGCCAAATGACCCAAACCGAAATGTAGATGAAAATGCTAATGCCAACAGTGCTGT AA AAAATAATAATAACGAAGAACCAAGTGATAAGCACATAAAAGAATATTTAAACAAAATACAAAATTCT CT TTCAACTGAATGGTCCCCATGTAGTGTAACTTGTGGAAATGGTATTCAAGTTAGAATAAAGCCTGGCT CT GCTAATAAACCTAAAGACGAATTAGATTATGCAAATGATATTGAAAAAAAAATTTGTAAAATGGAAAA AT GTTCCAGTGTGTTTAATGTCGTAAATAGTTCAATAGGATTAATAATGGTATTATCCTTCTTGTTCCTT AA TTAG 5′ BamHI Pf3D7 E/L pro CSP (SEQ ID NO: F12) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGATGAGAAA ATTAGCTATTTTATCTGTTTCTTCCTTTTTATTTGTTGAGGCC 3′ XhoI Pf3D7 CSP (5′ to 3′) (SEQ ID NO: F13) TCGCATCTCGAGCTAATTAAGGAACAAGAAGGATAATACC 3′ PstI Pf3D7 CSP (SEQ ID NO: F14) TCGCATCTGCAGCTAATTAAGGAACAAGAAGGATAATACC 5′ T5NT CSP repair (SEQ ID NO: F15) GCTATTCTATCTGTCTCTTCCTTCTTATTCGTTGAGGCC 3′ T5NT CSP repair (SEQ ID NO: F16) GGCCTCAACGAATAAGAAGGAAGAGACAGATAGAATAGC 5′ Loop out GPI CSP (SEQ ID NO: F17) GAAAAAAAAATTTGTAAAATGGAATAGCTGCAGtattatattttttatctaaaaaac 3′ Loop out CSP (SEQ ID NO: F18) GATAAAAAATATAATACTGCAGCTATTCCATTTTACAAATTTTTTTTTCAATATCATTTGC gi|124513463; XM_001350052.1; Plasmodium falciparum 3D7,  Thrombospondin-related anonymous protein, TRAP (TRAP) mRNA, complete cds (SEQ ID NO: F18) ATGAATCATCTTGGGAATGTTAAATATTTAGTCATTGTGTTTTTGATTTTCTTTGATTTGTTTCTAGT TAATGGTAGAGATGTGCAAAACAATATAGTGGATGAAATAAAATATCGTGAAGAAGTATGTAATGATG AGGTAGATCTTTACCTTCTAATGGATTGTTCTGGAAGTATACGTCGTCATAATTGGGTGAACCATGCA GTACCTCTAGCTATGAAATTGATACAACAATTAAATCTTAATGATAATGCAATTCACTTATATGCTAG TGTTTTTTCAAACAATGCAAGAGAAATTATTAGATTACATAGTGATGCATCTAAAAACAAAGAGAAGG CTTTAATTATTATAAAGTCACTCTTAAGTACAAATCTTCCATATGGTAAAACAAACTTAACTGATGCA CTGTTACAAGTAAGAAAACATTTAAATGACCGAATCAATAGAGAGAATGCTAATCAATTAGTTGTTAT ATTAACAGATGGAATTCCAGATAGTATTCAAGATTCATTAAAAGAATCAAGAAAATTAAGTGATCGTG GTGTTAAAATAGCTGTTTTTGGTATTGGACAAGGTATTAATGTAGCTTTCAACAGATTTCTTGTAGGT TGTCATCCATCAGATGGTAAATGTAACTTGTATGCTGATTCTGCATGGGAAAATGTAAAAAATGTTAT CGGACCCTTTATGAAGGCTGTTTGTGTTGAAGTAGAAAAAACAGCAAGTTGTGGTGTTTGGGACGAAT GGTCTCCATGTAGTGTAACTTGTGGTAAAGGTACCAGGTCAAGAAAAAGAGAAATCTTACACGAAGGA TGTACAAGTGAATTACAAGAACAATGTGAAGAAGAAAGATGTCTTCCAAAACGGGAACCATTAGATGT TCCAGATGAACCCGAAGATGATCAACCTAGACCAAGAGGAGATAATTTTGCTGTCGAAAAACCAAACG AAAATATAATAGATAATAATCCACAAGAACCTTCACCAAATCCAGAAGAAGGAAAGGGTGAAAATCCA AACGGATTTGATTTAGATGAAAATCCAGAAAATCCACCAAATCCACCAAATCCACCAAATCCACCAAA TCCACCAAATCCACCAAATCCAGATATTCCTGAACAAGAACCAAATATACCTGAAGATTCAGAAAAAG AAGTACCTTCTGATGTTCCAAAAAATCCAGAAGACGATCGAGAAGAAAACTTTGATATTCCAAAGAAA CCCGAAAATAAGCACGATAATCAAAATAATTTACCAAATGATAAAAGTGATAGATATATTCCATATTC ACCATTATCTCCAAAAGTTTTGGATAATGAAAGGAAACAAAGTGACCCCCAAAGTCAAGATAATAATG GAAATAGGCACGTACCTAATAGTGAAGATAGAGAAACACGTCCACATGGTAGAAATAATGAAAATAGA TCATACAATAGAAAACATAACAATACTCCAAAACATCCTGAAAGGGAAGAACATGAAAAGCCAGATAA TAATAAAAAAAAAGCAGGATCAGATAATAAATATAAAATTGCAGGTGGAATAGCTGGAGGATTAGCTT TACTCGCATGTGCTGGACTTGCTTATAAATTCGTAGTACCAGGAGCAGCAACACCCTATGCCGGAGAA CCTGCACCTTTTGATGAAACATTAGGTGAAGAAGATAAAGATTTGGACGAACCTGAACAATTCAGATT ACCTGAAGAAAACGAGTGGAATTAA 5′ BamHI Pf3D7 TRAP (SEQ ID NO: F19) GCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATCATCTTG GGAATGTTAAATATTTAGTCATTG 3′ XhoI Pf3D7 TRAP (5′ to 3′) (SEQ ID NO: F20) GCGCATCTCGAGTTAATTCCACTCGTTTTCTTCAGG Plasmodium falciparum 3D7 STARP antigen (PF07_9006) mRNA, complete cds  NCBI Reference Sequence: XM_001348922.1  gi|124511649|ref|XM_001348922.1| (PF07_0006) mRNA, complete cds (SEQ ID NO: F21) ATGATACATATTTTTTATAGGACAGCCATATTTACTCTCTCAATCTGGACAACACTGTTATATTCTAATA AAAATTTAAAATGTAATTTTTATTATAATAACAACAACTTATCAACATACGTTATAAAGCATAACAGATT TTTATCAGAATATCAATCGAACTTTCTTGGTGGGGGATATAGTGCAGCTTTAAAATTAGTAAATAGTAAA AAATCCGGAACAAATGTAAATACAAAGTATAATTCAGAAAATACCAATACAAATAATAATATACCAGAAA GTAGTAGTACATATACAAATACAAGGTTAGCAGCAAATAACAGTACAACTACAAGCACTACAAAAGTAAC AGATAATAATAAAACAAATATTAAATTAACAGGAAACAATAGTACAACTATAAATACAAATTCAACAGAA AATACTAGTGCTACCAAAAAAGTAACCGAAAATGTTATTACAAATCAAATATTAACAGGAAATAACAATA CAACCACAAATACATCCACGACAGAACATAATAATAATATTAACACAAATACAAATTCAACAGAAAATAC TAGTGCTACCAAAAAAGTAACCGAAAATGTTATTACAAATCAAATATTAACAGGAAATAACAATACAACC ACAAATACATCCACGACAGAACATAATAATAATATTAACACAAATACAAATTCAACAGATAATAGTAATA CTAATACAAATTTAACCGATAATACTTCTACAACTAAAAAGTTGACTGATAATATAAACACAACACAAAA TTTAACAACAAGTACTAATACAACTACAGTATCAACCGATAATAATAATATAAATACAAAACCCATTGAT AATAATAACACAGATATAAAATCGACAGATAATTATAACACAGGCACAAAGGAAACAGATAATAAGAACA CAGACATAAAAGCAACAGACAATAATAATATTACAACAACCACGGATAATACTAATACAAATGTAATATC AACAGATAATAGTAAAACAAATGTAATATCAACAGATAATAGTAAAACAAATACAATATCAACAGATAAT GATAATGCAGATACAATATTAACAGATAATGATAATAATACAGATATAATATTAACAGATAATAATAATA CAGATACAATATCAACAGATAATGATAATGCAGATACAAAAGCAACAGATAATAATAATAATACAAATAC AAAAGCAACAGATAATAATAATACAAAAATAATATCACCAGATAATAATAATACAAAAACAACATCAACA GATAATAATAATAATACAAATACAAAAGCAACAGATAATAATAATACAAAAACAATATCAAACGATAATA ATAATACAAAAACAATATCAACAGATAATAATAATACAAAAACAATATCAAACGATAATAATAATACAAA TACAATATCAACAGATAATAATAATAATAATACAAACCAATATGTCTTTGCTAACAATTATAATGAAACA ACTTCTGATGATGAACTAAATAAAGATTCCTGTGATTATTCAGAAGAAAAAGAAAATATAAAATCAATGA TTAACGCTTATTTAGACAAGTTAGATTTAGAAACTGTTCGTAAAATACATTCAGATATAAGTACATGTAT TGAAAAAAAAAATAATCCTAGGAATCAAATAACACATTTAAACAATTTAAAAAATATGTATAATATAATT AAATTTATAGTGGTTATATATATTGCTTTTAATTGGAGTGAAGTAATATATAAATATGTAGGAAAATTAA TTTTAGCTTTTGCTTTATATATGTTAATTAATTAA 5′ BamHI Pf3D7 E/L pro STARP (SEQ ID NO: F22)

3′ PstI Pf3D7 STARP (5′ to 3′) (SEQ ID NO: F23) TCGCATCTGCAGTTAATTAATTAACATATATAAAGCAAAAGCTAAAATTAATTTTCC gi|124802763|ref|XM_001347551.1| Plasmodium falciparum 3D7 25 kDa ookinete surface antigen precursor (pfs25) (PF10_0303) mRNA, complete cds ATGAATAAACTTTACAGTTTGTTTCTTTTCCTTTTCATTCAACTTAGCATAAAATATAATAATGCGAAAG TTACCGTGGATACTGTATGCAAAAGAGGATTCTTAATTCAGATGAGTGGTCATTTGGAATGTAAATGTGA AAATGATTTGGTGTTAGTAAATGAAGAAACATGTGAAGAAAAAGTTCTGAAATGTGACGAAAAGACTGTA AATAAACCATGTGGAGATTTTTCCAAATGTATTAAAATAGATGGAAATCCCGTTTCATACGCTTGTAAAT GTAATCTTGGATATGATATGGTAAATAATGTTTGTATACCAAATGAATGTAAGAATGTAACTTGTGGTAA CGGTAAATGTATATTAGATACAAGCAATCCTGTTAAAACTGGAGTTTGCTCATGTAATATAGGCAAAGTT CCCAATGTACAAGATCAAAATAAATGTTCAAAAGATGGAGAAACCAAATGCTCATTAAAATGCTTAAAAG AAAATGAAACCTGTAAAGCTGTTGATGGAATTTATAAATGTGATTGTAAAGATGGATTTATAATAGATAA TGAAAGCTCTATATGTACTGCTTTCTCAGCATATAATATTTTAAATCTAAGCATTATGTTTATACTATTT TCAGTATGCTTCTTCATAATGTAA 5′ EcoRI Synpro Pfs25 (SEQ ID NO: F25) ATGCGCATGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATAAACTTTACAGT TTGTTTCTTTTCCTTTTCATTCAACTTAGCATAAAATATAATAATGCGAAAGTTACCGTGGATACTGTATGCAAA AGAGGATTCTTAATTCAGATGAGTGG (176) 3′ SbfI Pfs25 (SEQ ID NO: F26) ATGCGCATCCTGCAGGTTACATTATGAAGAAGCATACTGAAAATAGTATAAACATAATGC gi|124802759; XM_001347550.1; Plasmodium falciparum 3D7 28 kDa ookinete surface protein (PF10_0302) mRNA, complete cds (SEQ ID NO: F27) ATGAATACATATTTCAAGGTACTACTGTTCCTATTCATCCAACTTTACATAACGTTGAATAAGGCTCGGG TTACTGAAAATACAATATGTAAATATGGATATCTAATCCAGATGAGTAATCATTATGAATGTAAGTGTAT TGAAGGATATGTATTAATAAATGAGGACACGTGTGGAAAAAAAGTAGTCTGTGATAAAGTTGAAAATTCA TTTAAAGCTTGTGATGAATACGCTTACTGTTTCGATTTAGGAAATAAGAATAATGAAAAACAGATAAAAT GTATGTGCAGAACAGAATATACTTTAACTGCTGGAGTATGTGTTCCTAATGTTTGTCGAGATAAAGTATG TGGTAAAGGAAAATGTATAGTAGATCCTGCAAATTCTTTAACACATACATGCTCATGCAATATAGGTACC ATACTTAACCAGAATAAATTATGTGATATACAAGGTGATACACCATGTTCATTAAAATGTGCAGAAAATG AAGTGTGTACATTAGAAGGAAATTATTATACATGTAAAGAAGATCCTTCATCTAACGGAGGAGGAAATAC TGTGGACCAGGCTGATACATCATATAGTGTAATAAACGGAGTAACCCTAACACACGTACTGATCGTATGC TCAATATTCATCAAATTGTTAATATAA 5′ BamHI Synpro (SEQ ID NO: F28) Pfs28ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATACATATT TCAAGGTACTACTGTTCCTATTCATCCAACTTTACATAACGTTGAATAAGGCTCGGGTTACTGAAAATACAATAT GTAAATATGG 3′PstI Pfs28 (SEQ ID NO: F29) ATGCGCATCTGCAGTTATATTAACAATTTGATGAATATTGAGCATACGATCAGTACG gi|124513649|ref|XM_001350145.1| Plasmodium falciparum 3D7 transmission blocking target antigen precursor (pfs45-48) mRNA, complete cds (SEQ ID NO: F29) ATGATGTTATATATTTCTGCGAAAAAGGCTCAAGTTGCTTTTATCTTATATATAGTATTAGTATTAAGAA TAATAAGTGGAAACAATGACTTCTGTAAGCCTAGCTCTTTGAATAGTGAAATATCTGGATTCATAGGATA TAAGTGTAATTTTTCAAATGAAGGTGTTCATAATTTAAAGCCAGATATGCGTGAACGTAGGTCTATCTTC TGCACCATCCATTCGTATTTTATATATGATAAGATAAGATTAATAATACCTAAAAAAAGTTCGTCTCCTG AGTTTAAAATATTACCAGAAAAATGTTTTCAAAAAGTATATACTGATTATGAGAATAGAGTTGAAACTGA TATATCGGAATTAGGTTTAATTGAATATGAAATAGAAGAAAATGATACAAACCCTAATTATAATGAAAGG ACAATAACTATATCTCCATTTAGTCCAAAAGACATTGAATTCTTCTGCTTCTGCGATAATACTGAAAAGG TTATATCAAGTATAGAAGGGAGAAGTGCTATGGTACATGTACGTGTATTAAAATATCCACATAATATTTT ATTTACTAATTTAACAAATGATCTTTTTACATATTTGCCGAAAACATATAATGAATCTAATTTTGTAAGT AATGTATTAGAAGTAGAATTGAATGATGGAGAATTATTTGTTTTAGCTTGTGAACTAATTAATAAAAAAT GTTTTCAAGAAGGAAAAGAAAAAGCCTTATATAAAAGTAATAAAATAATTTATCATAAAAACTTAACTAT CTTTAAAGCTCCATTTTATGTTACATCAAAAGATGTTAATACAGAATGTACATGCAAATTTAAAAATAAT AATTATAAAATAGTTTTAAAACCAAAATATGAAAAAAAAGTCATACACGGATGTAACTTCTCTTCAAATG TTAGTTCTAAACATACTTTTACAGATAGTTTAGATATTTCTTTAGTTGATGATAGTGCACATATTTCATG TAACGTACATTTGTCTGAACCAAAATATAATCATTTGGTAGGTTTAAATTGTCCTGGTGATATTATACCA GATTGCTTCTTCCAAGTATATCAACCTGAATCAGAAGAACTTGAACCATCCAACATTGTTTATTTAGATT CACAAATAAATATAGGAGATATTGAATATTATGAAGATGCTGAAGGAGATGATAAAATTAAATTATTTGG TATAGTTGGAAGTATACCAAAAACGACATCTTTTACTTGTATATGTAAGAAGGATAAAAAAAGTGCTTAT ATGACAGTTACTATAGATTCAGCATATTATGGATTTTTGGCTAAAACATTTATATTCCTAATTGTAGCAA TATTATTATATATTTAG 5′BamHI Synpro Pfs48.45 (SEQ ID NO: F30) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGATGTTATATATTTCT GCGAAAAAGGCTCAAGTTGCTTTTATCTTATATATAGTATTAGTATTAAGAATAATAAGTGGAAACAATGACTTC TGTAAGCCTAGCTCTTTGAATAGTGAAATATCTGG (185) 3′Pfs48.45 2ndT5NT (SEQ ID NO: F31) ATACGAATGGATGGTGCAGAAGATAGACCTACGTTCACGCATATCTGGCTTTAAATTATG (60) 5′Pfs48.45 3rdT5NT (SEQ ID NO: F32) ACCATCCATTCGTATTTTATATATGATAAGATAAGATTAATAATACCTAAAAAAAGTTCG (60) 3′Pfs48.45 3rdT5NT (SEQ ID NO: F33) TATAACCTTTTCAGTATTATCGCAGAAGCAGAAGAATTCAATGTCTTTTGGACTAAATGG (60) 5′Pfs48.45 4thT5NT (SEQ ID NO: F34) GATAATACTGAAAAGGTTATATCAAGTATAGAAGGGAGAAGTGCTATGGTACATGTACG (59) 3′Pfs48.45 4thT5NT (SEQ ID NO: F35) TTCAGGTTGATATACTTGGAAGAAGCAATCTGGTATAATATCACCAGGACAATTTAAACC (60) 5′Pfs48.45 END (SEQ ID NO: F36) CAAGTATATCAACCTGAATCAGAAGAACTTGAACCATCC (39)  3′Pfs48.45 (SEQ ID NO: F37) ATGCGCATCCTGCAGGCTAAATATATAATAATATTGCTACAATTAGGAATATAAATG (57) gi|124513579; XM_001350110.1; Plasmodium falciparum 3D7 sporozoite microneme protein (SPECT) (MAL13P1.212) mRNA, complete cds (SEQ ID NO: F38) ATGAAAATGAAAATCCCGATTTGTTTTCTCATTATTTTAGTCTTGTTAAAATGTGTGCTATCTTACAA TC TAAATAACGACTTATCAAAAAATAATAATTTTTCCTTAAATACATATGTCAGAAAAGATGATGTGGAA GA TGATTCAAAAAACGAGATTGTTGATAATATACAAAAAATGGTTGATGATTTTAGTGATGATATAGGTT TT GTAAAAACATCGATGCGTGAAGTTTTACTAGATACCGAAGCGTCCCTTGAAGAAGTATCAGATCATGT TG TACAAAACATATCAAAATATAGTTTAACCATTGAAGAGAAACTTAATCTTTTTGATGGGCTTCTTGAA GA ATTTATTGAAAATAATAAGGGCCTGATATCCAACTTATCAAAAAGACAACAAAAACTTAAGGGGGATA AA ATTAAAAAGGTTTGTGATTTGATCTTAAAAAAATTAAAAAAGTTAGAAAATGTCAACAAACTTATTAA AT ATAAGATAATATTAAAATATGGAAATAAAGATAATAAAAAAGAAATGATACAAACATTGAAAAATGAG GA GGGTTTATCTGATGACTTCAAAAATAATTTATCAAATTATGAAACAGAACAAAATAACGATGATATAA AA GAAATAGAATTAGTTAATTTTATTTCAACAAATTATGATAAGTTTGTTGTTAATCTAGAAGACCTTAA TA AGGAGTTGCTAAAGGATTTAAACATGGCCTTATCATAA 5′ BamHI Pf3D7 E/L pro SPECT (SEQ ID NO: F39) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAAATGAA AATCCCGATTTGTTTTCTC 3′ PstI Pf3D7 SPECT (SEQ ID NO: F40) TCGCATCTGCAGTTATGATAAGGCCATGTTTAAATCC gi|124505318; XM_001351365.1| Plasmodium falciparum 3D7 MAC/Perforin, putative (PFD0430c) mRNA, complete cds (SEQ ID NO: F41) ATGAAGTTACGTATTCTAAAAAAACACTATTACGTGGTCTTTATATTGTTGTATTTATATGACATTAGTT GTTTTAAATGTATACGTTTGAATAATCGTAGTATTTATAAGAATAAATACAAGAATAATGTCCATATAGG AACTAATGAGAATATAAGAAGTATAGAAAAATATTCCAATGTTTTATGTAATAGTATATTATGTAAAAAT GATAAAATTAGTTCATTTATAAATCAAAGAAAAAATGTAGATGATGATGATGAAAGTGAAAATGATGATA TGTATGAATCTACTACAGCTGGGAGTTCATCTGAAACTGATAACGAATCTGATGAAGAAGAAAATGATAG TAGTGACAATAATAATTCTGATGAAGAACAAATTGAAAATAGTAACAATAATAATTCTGATGAAGAACAA AATGACAGTAGTAGTAATGATAATAATGATGAGGAAAATGAGGAACAAGATGATGTTATGGATAATGATC AAAATGATAAAAAAATAAAACATTCATTCAATTTAGCAAACGAAAGCAAACATACAAAAGAAGAAAGAGT GAAGGAAGAAAAAAAATTAAAAATATACGATTTCATAAATGATAAAGAAAAAAGATTAAATTTTAATGGA GACCAAAAAGATGAAGATAATGAAGAAAATGATGATAAGGATGAAAATACGTTAGAAAATAGAAATATCA TAAGTAAACACACATCAGTATTTCCAGGTTTATATTTTATAGGTATAGGTTATAATTTACTTTTTGGTAA TCCTTTAGGAGAGGCTGATTCATTAATTGATCCAGGATATCGAGCTCAAATTTATTTAATGGAATGGGCT TTAAGTAAAGAAGGAATAGCAAATGACTTATCAACATTGCAACCAGTAAATGGATGGATAAGAAAAGAAA ATGCTTGTAGTAGAGTTGAATCGATCACCGAATGTTCTAGCATTTCAGATTATACTAAAAGTTTATCTGC AGAAGCTAAAGTATCAGGTTCTTATTGGGGTATTGCCTCTTTTTCTGCCTCAACAGGATATAGTAGTTTT CTTCATGAAGTTACCAAAAGGTCTAAGAAGACTTTTTTAGTTAAATCGAATTGTGTAAAATATACTATTG GACTTCCACCTTATATTCCATGGGATAAAACTACGGCTTATAAAAATGCCGTGAATGAACTACCCGCTGT CTTCACAGGGCTAGATAAAGAAAGTGAATGTCCTTCAGATGTTTATGAAGAAAATAAAACAAAAAGTAAT TGTGAAAATGTTAGTTTATGGATGAAATTTTTTGATATATATGGAACACACATTATTTACGAATCACAAT TAGGGGGAAAAATTACAAAAATTATAAACGTAAGCACTTCATCTATTGAACAAATGAAAAAAAATGGAGT TAGCGTAAAAGCAAAAATTCAAGCTCAGTTTGGTTTTGGAAGTGCTGGAGGATCTACTGATGTAAACTCT AGTAATTCCTCAGCAAATGATGAACAGAGTTATGATATGAATGAACAATTAATTGTGATTGGAGGAAATC CAATTAAGGATGTTACAAAAGAAGAAAATTTATTTGAATGGTCTAAAACGGTTACTAATCATCCTATGCC TATAAATATAAAATTAACACCTATATCAGATAGTTTTGATTCTGATGATTTAAAGGAATCTTATGACAAG GCTATTATATATTATTCAAGATTATATGGATTATCTCCCCATGATACTATGCAAAAGGATGATAAGGACA TAATCAAAATATTAACGAATGCCGATACAGTAACTAAGAATAGTGCTCCTCCCATCAATGCACAATGTCC ACATGGGAAAGTAGTTATGTTTGGTTTTTCCCTTAAACAAAATTTCTGGGATAACACGAATGCATTAAAG GGATATAACATAGAAGTATGTGAAGGAGGTTCTAATAGCTGTACCTCTAAGCAAGGAAGTTCCAATAAAT ATGATACGTCATATCTTTATATGGAATGTGGAGATCAGCCTTTGCCATTTTCAGAACAAGTGATCAGTGA AAGTACGTCAACATATAATACTGTAAAATGTCCCAATGATTACAGTATCCTTTTAGGGTTTGGTATATCC TCTTCCTCAGGGAGAATCAATTCAGCTGAGTATGTTTATTCTACTCCTTGCATACCAGGAATGAAAAGCT GCTCCTTAAATATGAATAACGATAACCAAAAGAGTTATATATACGTATTATGTGTTGACACTACCATATG GTCAGGAGTAAACAATTTATCACTTGTTGCTTTGGATGGAGCACATGGAAAAGTAAACAGATCAAAAAAA TATAGTGACGGAGAATTAGTAGGTACCTGTCCTTTAGATGGTACTGTATTGACAGGATTTAAAGTTGAAT TTCATACTTCAAGTCCATATGTACAAACACCATTTGAAAAATGTGCTAAAAGTTTAAAAGCCTGTTCTGT TCATGGTTCTGGACATGCTATAGGTATACAAAATTTCAAATCCTTATTTATTTATATGCTTTGCAAAAAT AATAAATGA  SPATR (SEQ ID NO: F42) ATGAAAAAAAGTCGTTTCTTGCTCCTCTCCATCTTCTTCTGTTTCGTAACAAATATAAGCTTGGAGTTCA AAAGAAAACAAAAAGTAGAAATTAATTCCTTACAAACAAATAAAAATAATGATAACATAAGAGAAGAAAA AATTAAAGGTGATGTGGAATCACAACCTGATGTTGATGGGGATACCTGCGTAATTTTTTCTTCTTCAGAA GGAAACTCCAGAAACTGTTGGTGCCCTAGAGGATACATTTTGTGCAGCGAAGAAGACGTTTTAGATGTAC AAGGCAAACTGAACGAAATTAAAAATAAGCATGAAAGAAGTCTAGTAACCCCTTTATGGATGAAGAGACT ATGTGACAATTCAAATGATGTAGGTTTTAAAAGTATGTCTGTTGTTATAGATTACGAATTAGCAGTATTA TGTAAAGACGGAAGTAATAAAGATTATGCTGATTTTGAAATTATTGGAGCATCTGGATATATTACAGGAG AAGAAATGATTGAAGAACAAAAAAGAAACCCTTGGTATGTTCCACGTAAATGTACTGTCAATAATTTTTA CTTGTGTAGAAAAGTAGAAAATGATAATGTCAATTGTTCATATACTCCTTGGTCAGATTGGAGTGCCTGT AAAAATAATACACAAAAAAGATATAGAAAAGTACGCCGATCCAATCAAAATAATGAAAATTTTTGTTTGT GGAATGACAAAATTGTTCCCAGAAATATAATGGAACAAACGCGTTCATGTTAA 5′EcoRT synpro SPATR (SEQ ID NO: F43) ATGCGCATGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAAAAAAGTCGTTTC TTGCTCCTCTCCATCTTCTTCTGTTTCGTAACAAATATAAGCTTGG (121)  PF3D7_1035700; duffy binding-like merozoite surface protein (DBLMSP) Previous ID(s): PF10_0348 (SEQ ID NO: F44) ATGAAGAAAATATATAGTATTTTCTTCTCTTTATTCATTTTGAATCTTCATATATATATAAAAAATATCAAATGC AATGACCTAATAAATTATAATGATTCGAATCTAAGAAACGGATTACTAAATAATAGTTTAGATTTAACAAATGGA TTAAATAACAAAGATAACAGTTTTATTGATTCTAAAATTGAAGAACATGAAAATAAATCTTACCAAAATAAAGAT AATAATATCTCTATCGTTGGACAAGATGTGCCTATTACATCGGTATATTCTTCTAAAATTATAAATGCTAATGAT TTAGAAGGAAATAGTATTGACGATACTAAAGGTCTTAGTGTTACTAATAGTGGATTTGATGATGGTAGTGCTTTT GGTGGTGGACTCCCTTTCTCTGGTTATTCTCCTCTACAAGGAAATCATAATAAATGTCCTGATGAAAATTTCTGT AAGGGTATTAAAAATGTCTTATCCTGTCCTCCAAAAAATTCTACTGGTAGAAATGGGGATTGGATTAGTGTGGCT GTTAAAGAAAGTTCAACTACAAATAAAGGTGTTCTTGTTCCCCCCAGAAGAACAAAATTATGTCTAAGAAATATT AACAAGGTTTGGCATCGAATCAAAGACGAGAAAAATTTTAAAGAAGAATTTGTTAAAGTTGCTTTAGGAGAATCA AATGCTTTAATGAAACATTATAAAGAAAAAAATCTGAATGCCCTTACAGCTATAAAATATGGATTTTCAGATATG GGAGATATAATAAAGGGAACAGACCTAATTGACTATCAAATTACTAAAAATATAAATAGGGCATTAGATAAAATA TTACGTAATGAAACAAGTAATGACAAAATTAAAAAACGTGTAGACTGGTGGGAAGCTAATAAAAGTGCATTCTGG GATGCATTCATGTGTGGATATAAAGTTCATATCGGAAATAAACCATGTCCAGAACATGATAATATGGACAGAATA CCACAATATCTTAGATGGTTTAGAGAATGGGGAACATATGTTTGCAGCGAATATAAAAATAAGTTTGAGGATGTA ATAAAATTATGTAATATCCAACAATTTACAAACCAGGATGATTCACAACTATTAGAAATATCAAAAAAGGATAAA TGTAAAGAAGCATTAAAGCATTATGAAGAATGGGTTAATAGAAGGAGACCTGAATGGAAAGGCCAATGTGATAAA TTTGAAAAAGAAAAAAGTAAATATGAAGATACTAAAAGTATAACTGCTGAAAAATATTTAAAAGAAATATGTTCT GAATGTGATTGTAAATATAAAGATTTGGATAATACATTTAAAGAATTTAAAGATAACGTTACACTTCTTAAAGCA GTAATTGATAACAAAAAAAATCAAGATTCTCTAACAACCACTTCTTTATCAACGTCTATTAATAGTGTTAGGGAT TCTAGTAATCTAGATCAACGAGGGAATATAACAACATCTCAAGGAAATTCACACCGTGCAACTGTTGTGCAACAA GTTGATCAAACCAACAGATTAGATAATGTAAACTCTGTAACGCAAAGAGGAAATAATAACTACAACAATAATTTA GAGCGTGGATTGGGTTCTGGTGCTCTTCCTGGTACAAATATTATTACTGAAGAAAAATATTCTCTAGAATTAATA AAATTAACATCAAAGGATGAAGAAGATATTATAAAGCATAATGAGGATGTGAGAGAAGAAATAGAAGAACAACAA GAAGACATCGAGGAAGATGAAGAAGAATTGGAAAATGAAGGAGAAGAAACAAAAGAAGAAGATGATGAAGAAAAG AATGAAACAAATGATACGGAAGATACGGACGATACTGAAGATACGGAAGATATAGAAGAGGAAAATAAGGAAAAA GAACTCAGTAATCAACAACAAAGTGAAAAAAAAAGTATTTCAAAAGTTGACGAAGATTCATATCGAATACTATCA GTAAGTTATAAGGACAATAATGAAGTAAAAAATGTTGCTGAATCTATAGTGAAAAAACTATTTAGTTTATTTAAT GATAATAATAATTTGGAAACTATTTTTAAGGGTTTGACAGAAGATATGACAGATTTATTTCAAAAATAA 5′ EcoRI Synpro DBLMSP (SEQ ID NO: F45) (SEQ ID NO: F46) ATGCGCATGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAGAAAATATATAGT ATTTTCTTCTCTTTATTCATTTTGAATCTTCATATATATATAAAAAATATCAAATGC (132)  3′ SbfI DBLMSP (SEQ ID NO: F47) GCGCATCCTGCAGGTTATTTTTGAAATAAATCTGTCATATCTTCTGTCAAACCC (54) Overlapping Primers to correct T5NT's: 5′ WPP DBLMSP T5NTs (SEQ ID NO: F48) GGATTTGATGATGGTAGTGCTTTTGGTGGTGGACTCCCTTTCTCTGGTTATTCTCCTCTACAAGGAAATCATAAT AAATGTCCTGATGAAAATTTCTGTAAGGGTATTAAAAATGTCTTATCCTGTCCTCC (131) 3′ WPP DBLMSP T5NTs (SEQ ID NO: F49) GGACAGGATAAGACATTTTTAATACCCTTACAGAAATTTTCATCAGGACATTTATTATGA (60)  gi|1699002|GENBANK: U78724.1 and PFU78724, Plasmodium falciparum erythrocyte binding antigen region II (EBA-175) gene, partial cds (SEQ ID NO: F50) ATGGGAAGAAATACTTCATCTAATAACGAAGTTTTAAGTAATTGTAGGGAAAAAAGGAAAGGAATGAAATGGGA TTGTAAAAAGAAAAATGATAGAAGCAACTATGTATGTATTCCTGATCGTAGAATCCAATTATGCATTGTTAATCT TAGCATTATTAAAACATATACAAAAGAGACCATGAAGGATCATTTCATTGAAGCCTCTAAAAAAGAATCTCAACT TTTGCTTAAAAAAAATGATAACAAATATAATTCTAAATTTTGTAATGATTTGAAGAATAGTTTTTTAGATTATGG ACATCTTGCTATGGGAAATGATATGGATTTTGGAGGTTATTCAACTAAGGCAGAAAACAAAATTCAAGAAGTTTT TAAAGGGGCTCATGGGGAAATAAGTGAACATAAAATTAAAAATTTTAGAAAAAAATGGTGGAATGAATTTAGAGA GAAACTTTGGGAAGCTATGTTATCTGAGCATAAAAATAATATAAATAATTGTAAAAATATTCCCCAAGAAGAATT ACAAATTACTCAATGGATAAAAGAATGGCATGGAGAATTTTTGCTTGAAAGAGATAATAGATCAAAATTGCCAAA AAGTAAATGTAAAAATAATACATTATATGAAGCATGTGAGAAGGAATGTATTGATCCATGTATGAAATATAGAGA TTGGATTATTAGAAGTAAATTTGAATGGCATACGTTATCGAAAGAATATGAAACTCAAAAAGTTCCAAAGGAAAA TGCGGAAAATTATTTAATCAAAATTTCAGAAAACAAGAATGATGCTAAAGTAAGTTTATTATTGAATAATTGTGA TGCTGAATATTCAAAATATTGTGATTGTAAACATACTACTACTCTCGTTAAAAGCGTTTTAAATGGTAACGACAA TACAATTAAGGAAAAGCGTGAACATATTGATTTAGATGACTTCTCTAAATTTGGATGTGATAAAAATTCCGTTGA TACAAACACAAAGGTGTGGGAATGTAAAAAACCTTATAAATTATCCACTAAAGATGTATGTGTACCTCCGAGGAG GCAAGAATTATGTCTTGGAAACATTGATAGAATATACGATAAAAACCTATTAATGATAAAAGAGCATATTCTTGC TATTGCAATATATGAATCAAGAATATTGAAACGAAAATATAAGAATAAAGATGATAAAGAAGTTTGTAAAATCAT AAATAAAACTTTCGCTGATATAAGAGATATTATAGGAGGTACTGATTATTGGAATGATTTGAGCAATAGAAAATT AGTAGGAAAAATTAACACAAATTCAAATTATGTTCACAGGAATAAACAAAATGATAAGCTTTTTCGTGATGAGTG GTGGAAAGTTATTAAAAAAGATGTATGGAATGTGATATCATGGGTATTCAAGGATAAAACTGTTTGTAAAGAAGA TGATATTGAAAATATACCACAATTCTTCAGATGGTTTAGTGAATGGGGTGATGATTATTGCCAGGATAAAACAAA AATGATAGAGACTCTGAAGGTTGAATGCAAAGAAAAACCTTGTGAAGATGACAATTGTAAACGTAAATGTAATTC ATATAAAGAATGGATATCAAAAAAAAAAGAAGAGTATAATAAACAAGCCAAACAATACCAAGAATATCAAAAAGG AAATAATTACAAAATGTATTCTGAATTTAAATCTATAAAACCAGAAGTTTATTTAAAGAAATACTCGGAAAAATG TTCTAACCTAAATTTCGAAGATGAATTTAAGGAAGAATTACATTCAGATTATAAAAATAAATGTACGATGTGTCC AGAAGTAAAGGATGTACCAATTTCTATAATAAGAAATAATGAACAAACTTCGTAA 5′ BAM synpro175RII (SEQ ID NO: F51) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGGGAAGAAAT ACTTCATCTAATAACG 3′ pst175RII (SEQ ID NO: F52) GCGCATCTGCAGTTACGAAGTTTGTTCATTATTTCTTATTATAGAAATTGGTACATCC 5′ 175RIIWPP (SEQ ID NO: F53) CGTGAACATATTGATTTAGATGACTTCTCTAAATTTGGATGTGATAAAAATTCCG 3′ 175RIIWPP (SEQ ID NO: F54) CGGAATTTTTATCACATCCAAATTTAGAGAAGTCATCTAAATCAATATGTTCACGC 5′ EBARII T5NT (SEQ ID NO: F55) GCGTGAACATATTGATTTAGATGACTTCTCTAAATTTGGATGTGATAAAAATTCCG 3′ EBARII T5NT (SEQ ID NO: F56) GTGTTTGTATCAACGGAATTTTTATCACATCCAAATTTAGAGAAGTCATCTAAATC 5′ EBARII T5NT SEQ  (SEQ ID NO: F57) GCTAAAGTAAGTTTATTATTGAATAATTGTGATGCTGAATATTCAAAATATTGTGATTG 5′ EbaRII T5NT NEW (SEQ ID NO: F58) GATTTAGATGACTTCTCTAAATTTGGATGTGATAAAAATTCCGTTGATACAAACAC 3′ EbaRII T5NT NEW (SEQ ID NO: F59) CGGAATTTTTATCACATCCAAATTTAGAGAAGTCATCTAAATCAATATGTTCACGCTTTT MSP1-42 (SEQ ID NO: F60) ATGGCAATATCTGTCACAATGGATAATATCCTCTCAGGATTTGAAAATGAATATGATGTTATATATTT AAAACCTTTAGCTGGAGTATATAGAAGCTTAAAAAAACAAATTGAAAAAAACATTTTTACATTTAATT TAAATTTGAACGATATCTTAAATTCACGTCTTAAGAAACGAAAATATTTCTTAGATGTATTAGAATCT GATTTAATGCAATTTAAACATATATCCTCAAATGAATACATTATTGAAGATTCATTTAAATTATTGAA TTCAGAACAAAAAAACACACTTTTAAAAAGTTACAAATATATAAAAGAATCAGTAGAAAATGATATTA AATTTGCACAGGAAGGTATAAGTTATTATGAAAAGGTTTTAGCGAAATATAAGGATGATTTAGAATCA ATTAAAAAAGTTATCAAAGAAGAAAAGGAGAAGTTCCCATCATCACCACCAACAACACCTCCGTCACC AGCAAAAACAGACGAACAAAAGAAGGAAAGTAAGTTCCTTCCATTTTTAACAAACATTGAGACCTTAT ACAATAACTTAGTTAATAAAATTGACGATTACTTAATTAACTTAAAGGCAAAGATTAACGATTGTAAT GTTGAAAAAGATGAAGCACATGTTAAAATAACTAAACTTAGTGATTTAAAAGCAATTGATGACAAAAT AGATCTTTTTAAAAACCCTTACGACTTCGAAGCAATTAAAAAATTGATAAATGATGATACGAAAAAAG ATATGCTTGGCAAATTACTTAGTACAGGATTAGTTCAAAATTTTCCTAATACAATAATATCAAAATTA ATTGAAGGAAAATTCCAAGATATGTTAAACATTTCACAACACCAATGCGTAAAAAAACAATGTCCAGA AAATTCTGGATGTTTCAGACATTTAGATGAAAGAGAAGAATGTAAATGTTTATTAAATTACAAACAAG AAGGTGATAAATGTGTTGAAAATCCAAATCCTACTTGTAACGAAAATAATGGTGGATGTGATGCAGAT GCCACATGTACCGAAGAAGATTCAGGTAGCAGCAGAAAGAAAATCACATGTGAATGTACTAAACCTGA TTCTTATCCACTTTTCGATGGTATTTTCTGCAGTTCCTCTAACTTCTTAGGAATATCATTCTTATTAA TACTCATGTTAATATTATACAGTTTCATTTAA 5′BAMHISYNPROMSP1-42 (SEQ ID NO: F61) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGGCAATATC TGTCACAATGGATAATATCCTCTC 3′SBFINSP1-42 (SEQ ID NO: F62) GCGCATCCTGCAGG TTAAATGAAACTGTATAATATTAACATGAGTATTAATAAGAATGATATTCC MSP1-19 (SEQ ID NO: F63) ATGTTAAACATTTCACAACACCAATGCGTAAAAAAACAATGTCCAGAAAATTCTGGATGTTTCAGACA TTTAGATGAAAGAGAAGAATGTAAATGTTTATTAAATTACAAACAAGAAGGTGATAAATGTGTTGAAA ATCCAAATCCTACTTGTAACGAAAATAATGGTGGATGTGATGCAGATGCCACATGTACCGAAGAAGAT TCAGGTAGCAGCAGAAAGAAAATCACATGTGAATGTACTAAACCTGATTCTTATCCACTTTTCGATGG TATTTTCTGCAGTTCCTCTAACTTCTTAGGAATATCATTCTTATTAATACTCATGTTAATATTATACA GTTTCATT TAA 5′BAMHI SYNPRO MSP1-19 (SEQ ID NO: F64) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGTTAAACAT TTCACAACACC 3′ SBFIMSP1-19 (SEQ ID NO: F65) GCGCATCCTGCAGGTTAAATGAAACTG SIAP1 (SEQ ID NO: F66) ATGGAAGGCTTTGTTGCTTTGCTGTCTTTCCTGGTGGTGTTGGTTTTTAATAAAACTATAGGATATAACATAAAA TCTGGGAACACATCAAATAATATAAAATATGTAAATGTGTTAGATAATGATAGAGATATAAATACACATAGTGTA TTACCCGAAGTAGAAAATGTGATAGAGAGAAAGGATATTTATAGACAAATAAATTTTATGGAAACGTTTGTATCT AGTAATAATATGATGCACGATAGAGAGAAACATACATCTAATGATTCAGGTTCTTATGAAATTACAGGTATAGTT GATGGTATGAAAATAGGACATCCGATATCGGTTGCTTTAGGTTCTCAATATTCTAATTATTTTGATTATTTACAA ATAGTACATTTAGATTACACAAATTCACGTTTTAGTTTTACTGTTGGTGAAGGTAAATATTATTTACGTACTTAT GGAAGTACTTATATGACACCTAGTGCTATAAAAATTAAAGTACCTTGTGAAAAATGTAAATTTATAAATTCTGAA TATAGTGGTATCATAAAAATTATTCCATATGAAACTAATAATAACTTATTTATTTATAATTGGGTATTACAAACA TCATCACCATTAGCTTTAGAAAATATAAATACAGTATTTAGTGATGAAGCAGATTTAATTCATGGAAATAGTTTA TCAGAGGAATTTAAAATAGATTCGTCAGCTGCAGCTACATCTTTAAATACATTTTATGGAATTGTATTACATGGT ATATGGAGCTCTGAATATGCCGAAAGATTATTAACCGTTATTTCTGAATTTCCAGATTGTGTAAAAATGTCTGCT CATGATAAAAATGCTAGATCGAAACAAAGAAAAAATCAAAAGTGGATTCTAGTTAATGAAGATTTAGGATCTTTT GATATGAAAATGGAAGTATGTGAAGAAGTTAATTGTGATTATTCTGCCATAATTCATGTTTCTAAACATGCTTTT GAATATTCTAAAAAGCTTGTTCATAACAGAGGTCGTAATGGAAGATACTATTCAAGAAGAGTTGAAAAAATTTTA ATAAGAGCTCTTTTATCATTAGATTTTTCTTTATTTATTACGTATTTTCAACAGAAACATGGTGTTACCTTATTA GATCCACAATATGATTATGAATTGATAACAAATATGTCTGGTTATTCATCTAATAATTATCAATCTTGGAATCAT AATTTGGAAGAACTTGTCGAATTAGCTACTTCATGGGATGAATATCCAAAAGGACTTCAAAAAGTACAAGGTTTA TCATATTTATTAAGAAGAAAAAATGGTACTAAACATCCAGTATATCCAACAGCACCAGCTGTAGCTTTTCCTGCT GGATCTCAAAATAATTCATTTATTGAATTTATGGAATCAGCATTTGTAAATTATGTAGATATATCACATCTAGTT ATTCATGAAGTGGCACATTTTATATGGGTTAATACCGTTTCAAAAGAATTAAAAGAAAAATGGATTCAAATCGGA CAATGGTATAAAGAACCTTTATCACCTAGTGAATGGGCTACAAAATTAGAAGTAGAATTTGTCTCAGCATATGCT CATGATAAAAATCCTGCAGAAGATTTTGCAGAATCTATGGCTACATATGTTTTAAATTCGAAATTATTAAATTCT AGATCTTTTGATAAATTCAAATGGATTCAAGATAACTTATTTGGTGGTGGATTTTATATTACAACTGGTACCCAC AAATTTGATGTTATTAATTTAGGTAATGAAGTATATTATTTCCCTGGAAAAGTTACAAGAGTACGTGCAAAGGTT CTAGGAAGTCCGACTGAAGATAAATTAGTTAAAATATATATTTCTTTATTATCTAGTGATGGTTCTGAAGGTTGT GCTAAACATGGTTATGCAAGAATATTTTCAGAACAACAAACTTTTAGAGATTTATATTTTCATACAGAAGATAGA TCACCATGTAGTCATAAATTATATGGAGAATTTACTATGAATAAACATGAAAGTAGAGGTAGATGGACAGCTGAA TCTATGATATTTACAGGAGAAAATAATATAGAAAGATATGTTGGTTTAGGATCCTTCCATTTTTATTTATATGTT AATAATCAAAATGAAGATGTAGAAAAACCAATTCCACTTTTAGATTCTATATCTATCTATACTCATAATGCTACA GAAACAAATGATGCTCTATTAAGATTACATGTGATGGTTTTAGAAAATGAATTAATAAAAGAACATGGTGGACCT TATGCTAGTTTTGCTGCTCATGAAAATAAAAGCTACTCATATGAAAGTCGTACATATAAAATGTATCCACCTGAA TTTAATACATTAATGTTAAAAGCAGATTATTTTATAAGAGATATAAATACACGAGGATTTAGAGAAGTAAATATG GATTCATGTAAATCATATACAAATATGGATACAAGAAATTTAAAATGTTTTCAAGTCTTAAATCCAGTTACTATT CCAAAATATTGCATAGGAAGCACATATTTCTTAAGACAAGTTTCCATTGAAGATATAGCAGGAAACCTAGAAACT GTAAATATCTCTTCAGATAAATATTCTGCTCGTTTACATCCTATAGGTGTACGAGATAAACAAAAACCGGTTGTA TCAAATGTAAGGGTTTCAAGTAAACCAGCTAATGAATATCATGATGGAGAAACCATTGTATCTCTAAGTTTTAAT GTTCATGATAATTTATCAGGAGTCTATTATATTTTTGTATATCTAAGAGATCCACATGGTGGAAAACACAGAAGT GATATTGATAGAGCATCCTTACCAACAGGTACAGAAAATAAGCAAATAAATCACAAAATCTTGTTACCAAAAGGT TCTATGGGTGGTACATGGATGTTAGAAGAAATCAAAGCAGTTGATTCATGTAAAAATGAATCTAGAAATATATAT ACTCATAGTGTTTACGTTCAAAATGATTAA 5′SIAP1 (5′primer) (SEQ ID NO: F67) GTATATTCAAAAAATGAGTTATATAAAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGGAA GGCTTTGTTGCTTTGCTGTCTTTCCTGGTGGTGTTGG (112bp) 3′SIAP1 5′A26L RRA (5′primer) (SEQ ID NO: F68) CTCATAGTGTTTACGTTCAAAATGATTAAATATCAGACTTCAGTATCCCAAGTAGC (56 bp)  SIAP2 (SEQ ID NO: F69) ATGAACATGTACGTAATCTATTACTACTTCTTAATCTTAATCTTCATAAATTCCTGCCATCTATACTT ATCTTCCGAAAACCAAAAAAAAACTATTGCTACTGTTCATAATAACACAAGAACGAATACATTAAAAA AAAATAATAGTAATAATAATAATACAAATGATATATTTGGTTTAAGCCCCTCTGAAGTACCAAATTTA ATAGATGATGAAGAAGAATATGAAATACTTGAAGGAGTCAAAAATAATTCTGATTTAACAAGATCAAC TAACATAGACCACCCCTCTCCTTCTTCTACTATGATAGACCTTGATAATATAATTACAGAAATCTCAA AATTAAAAAAAAAAAAATTAAAAAAAGAAATGAACAATAAATTAGAGCAACAAACAAATCAAAATAAC AATACTATTCATCATAATAATGAAAATGAAATAAATACCTTCACACAAAATATTAAAACAAATTCAAA TGAACTCAAAAATCAAGATTCATCAAATAGTCTTATTAGTACTAATAGTAATACGATGGATGAATTAT TGTTATACAGTACTAACTCAGAAGATAATTTAGATATTTCTTTTGGTGAACTTCAATTATATGAGAAC AGTGATGAAGATACAAGTGATTATGAATATGTTAATGAAGATTATTCCGTGAATCATATTTTTTCAAA TGATACAGAAGAATCATTTAATATTTTAGAAGATGTTGAAAATATATCATTATCATCAAGTAATAGAT ATCAATATAGTCCTATCGGACCATATAAAAAAAAACACACAAAGGTTTTTGAAAATTTTAAAATGACA AGAAATTATGACGAATTTCTTAAACTCTACAATTTAAAAGATTCTTCAGATAATCAAGAAGAATATTA TGAATTGTTAGCAGGAGAACCTTTTAAACTTAATAGTTACTATTATAGAGATGTAAAGTATGAAAATG TTAAAAAATATATATTTAAAGAAATTTATGATAATATTCAAAATATAAGTACAGAAAATAAAATAATT GTATCAAAAAAAGAGGAACTCTTCTTCTATTTCATCAAAAGTTTATTGAAAAATAATTTTATATGCTT ATCCTATGAAGAAGAGGAAAATTTATTGAGTGAATCTAAACTCTTACTAGAAGCTATGATATCTAAAA AAATACAATAA 5′SIAP2 (5′ primer) (SEQ ID NO: F70) GTAAAAATAAATCACTTTTTATACTAATAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA AAAATGAACATGTACGTAATCTATTACTACTTCTTAATCTTAATCTTCATAAATTCCTGCC (129 bp) 3′SIAP2 5′A56R RRA (5′ primer) (SEQ ID NO: F71) CTAGAAGCTATGATATCTAAAAAAATACAATAAATTTTTGACTTACATAAATGTCTGG (58 bp) gi|124505604|ref|XM_001351508.1| Plasmodium falciparum 3D7 reticulocyte- binding protein homologue 5 (PfRh5) mRNA, complete cds (SEQ ID NO: F72) ATGATAAGAATAAAAAAAAAATTAATTTTGACCATTATATATATTCATCTGTTTATATTAAATAGATTAAGTTTT GAAAATGCAATAAAAAAAACGAAGAATCAAGAAAATAATCTGACGTTACTACCAATAAAGAGCACTGAAGAAGAA AAAGATGATATAAAAAATGGAAAGGATATAAAAAAAGAAATTGATAATGATAAAGAGAATATAAAAACAAATAAT GCTAAAGATCATTCAACATATATAAAATCATATTTGAATACAAATGTAAATGATGGTTTAAAATATTTGTTTATT CCTTCTCATAATTCTTTTATAAAAAAATATTCTGTATTTAATCAAATAAATGATGGCATGTTATTAAATGAAAAA AATGATGTGAAAAATAATGAAGACTATAAAAATGTGGATTATAAAAATGTTAATTTCCTACAATATCATTTTAAA GAGTTATCAAATTATAACATTGCAAATTCTATTGATATTTTACAAGAAAAAGAAGGACATTTGGATTTTGTTATA ATACCTCATTATACTTTCCTAGATTATTATAAACATTTATCTTATAATTCTATATATCATAAGTCCTCTACATAT GGAAAGTGTATAGCTGTAGATGCTTTTATTAAGAAAATAAATGAAACATATGACAAAGTGAAAAGTAAATGTAAT GATATAAAGAATGATTTAATTGCAACTATAAAAAAATTAGAGCATCCTTATGATATAAATAATAAGAATGATGAT TCCTATAGATATGATATATC TGAAGAAATCGATGATAAATCTGAAGAGACAGATGATGAAACCGAAGAGGTAGAAGATAGTATACAAGATACAGA TAGTAATCATACTCCTTCAAATAAAAAAAAAAATGATCTTATGAATAGAACGTTTAAAAAGATGATGGATGAATA TAATACAAAAAAAAAAAAATTAATTAAATGTATAAAAAACCATGAGAATGATTTTAATAAAATATGTATGGATAT GAAAAATTATGGTACAAACCTTTTTGAACAACTTTCATGTTACAATAATAATTTCTGTAATACAAACGGAATAAG ATATCATTATGATGAATATATTCATAAATTAATATTATCTGTTAAATCAAAAAACTTAAATAAAGACCTATCAGA TATGACAAATATTTTACAACAAAGTGAATTATTATTAACCAATTTAAATAAAAAAATGGGTTCCTATATATATAT TGATACAATAAAATTTATACATAAAGAAATGAAACATATTTTTAACAGAATTGAATATCATACAAAAATAATAAA CGATAAAACTAAAATAATTCAAGACAAAATTAAATTAAATATATGGAGAACATTTCAAAAAGATGAATTATTAAA AAGAATTTTAGACATGTCAAATGAATATTCTTTATTTATTACTAGTGATCATTTAAGACAAATGTTATATAATAC ATTCTATTCAAAAGAAAAACATTTAAATAATATATTTCATCATTTAATTTATGTACTACAAATGAAGTTCAATGA TGTCCCAATTAAAATGGAAT ATTTTCAAACATATAAAAAAAATAAACCACTTACACAATGA 5′Bam synpro-Rh5 (SEQ ID NO: F73) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGATAAGAATAAAAAAAAAATTAAT TTTGACC  3′PstI-Rh5 (SEQ ID NO: F74) GCGCATCTGCAGTCATTGTGTAAGTGGTTTATTTTTTTTATATGTTTGAAAATATTCC  5′WPP-Rh5T5NT 1 (SEQ ID NO: F75) GGATTATAAAAATGTTAATTTCCTACAATATC 3′WPP-Rh5T5NT 1 (SEQ ID NO: F76) CTCTTTAAAATGATATTGTAGGAAATTAACATT 5′WPP-Rh5T5NT 2 (SEQ ID NO: F77) GTTATAATACCTCATTATACTTTCCTAGATTATTATAAAC 3′WPP-Rh5T5NT 2 (SEQ ID NO: F78) GATAAATGTTTATAATAATCTAGGAAAGTATAATGAGG 5′Bamsynpro-Rh5 (SEQ ID NO: F79) ATGCGCATGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGATAAGAATAAAAAAA AAATTAATTTTGACCATTATATATATTCATCTGTTTATATTAAATAGATTAAGTTTTGAAAATGCAATAAAAAAA ACGAAGAATCAAGAAAATAATCTGACGTTACTACCAATAAAGAGC 3′PstI-Rh5 (SEQ ID NO: F80) GCGCATCTGCAGTCATTGTGTAAGTGGTTTATTTTTTTTATATGTTTGAAAATATTCCATTTTAATTGGGACATC ATTGAACTTCATTTGTAGTACATAAATTAAATGATGAAATATATTATTTAAATGTTTTTCTTTTGAATAGAATG Plasmodium falciparum 3D7 sexual stage-specific protein precursor  (PFD0310w) mRNA, complete cds (SEQ ID NO: F81) ATGAATATTCGAAAGTTCATACCATCTTTAGCTTTAATGCTTATATTCTTCGCTTTTGCAAACCTGGTAT TATCAGATGCAAATGACAAAGCAAAAAAGCCCGCTGGAAAAGGATCCCCTTCAACTTTGCAAACCCCAGG AAGTTCTTCAGGTGCCTCTCTTCATGCTGTTGGACCTAATCAAGGTGGACTATCTCAAGGTCTTTCTGGA AAAGATTCTGCTGACAAAATGCCTTTAGAAACTCAGCTAGCTATAGAAGAAATCAAGAGCTTATCCAATA TGTTAGATAAAAAAACGACAGTTAACAGAAACTTAATCATAAGTACTGCTGTCACAAATATGATCATGTT GATCATATTATCTGGTATAGTTGGATTTAAAGTTAAAAAAACGAAGAACGCAGATGATGATAAAGGAGAT AAGGATAAGGACAAGGATAATACAGATGAAGGAGACGAAGGAGATGATTCTTAA 5′ EcoRI SYNPRO Pfs16 (SEQ ID NO: F82) AGTCGAGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAATATTCGAA AGTTCATACC 3′ PstI Pfs16 (SEQ ID NO: F83) CGTATTCTGCAGTTAAGAATCATCTCCTTCGTCTCC Plasmodium falciparum 3D7 Merozoite Surface Protein 9, MSP-9 (MSP-9) mRNA,  complete cds; gi|124806293|ref|XM_001350647.1 (SEQ ID NO: F84)

5′-MfeI synpro MSP9, P. falciparum (SEQ ID NO: F85) ATGCGCATCAATTGAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGATGAACATGAAAATT GTTTTATTCAGTTTATTGC  3′-SbfI MSP9 P. falciparum (SEQ ID NO: F86) GCGCATCCTGCAGGTTATTTTGATTCTTCAGTTGTCAAATTTTCTGGTAC PF3D7_1335100; merozoite surface protein 7 (MSP7); Previous ID(s):  PF13_0197, P. falciparum (SEQ ID NO: F87) ATGAAGAGTAATATCATATTTTATTTCTCTTTCTTCTTTGTGTACTTATACTATGTTTCGTGTAATCAATCAACT CATAGTACACCAGTAAATAATGAAGAAGATCAAGAAGAATTATATATTAAAAATAAAAAATTGGAAAAACTAAAA AATATAGTATCAGGAGATTTTGTTGGAAATTATAAAAATAATGAAGAATTATTAAACAAAAAAATTGAAGAATTA CAAAACAGTAAAGAAAAAAATGTACATGTATTAATTAATGGAAATTCAATTATTGATGAAATAGAAAAAAATGAA GAAAATGATGATAACGAAGAAAATAATGATGATGACAATACATATGAATTAGATATGAATGATGACACATTCTTA GGACAAAATAACGATTCACATTTTGAAAATGTTGATGATGACGCAGTAGAAAATGAACAAGAAGATGAAAACAAG GAAAAATCAGAATCATTTCCATTATTCCAAAATTTAGGATTATTCGGTAAAAACGTATTATCAAAGGTAAAGGCA CAAAGTGAAACAGATACTCAATCTAAAAATGAACAAGAGATATCAACACAAGGACAAGAAGTACAAAAACCAGCA CAAGGAGGAGAATCGACATTTCAAAAAGACCTAGATAAGAAATTATATAATTTAGGAGATGTTTTTAATCATGTA GTTGATATTTCAAACAAAAAGAACAAAATAAATCTCGATGAATATGGTAAAAAATATACAGATTTCAAAAAAGAA TATGAAGACTTCGTTTTAAATTCTAAAGAATATGATATAATCAAAAATCTAATAATTATGTTTGGTCAAGAAGAT AATAAGAGTAAAAATGGCAAAACGGATATTGTAAGTGAAGCTAAACATATGACTGAAATTTTCATAAAACTATTT AAAGATAAGGAATACCATGAACAATTTAAAAATTATATTTATGGTGTTTATAGTTATGCAAAACAAAATAGTCAC TTAAGTGAGAAAAAAATAAAACCAGAAGAGGAATATAAAAAATTCTTAGAATATTCATTTAATTTACTAAACACA ATGTAA  5′EcoRI Synpro MSP7 (SEQ ID NO: F87) ATGCGCATGAATTCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAGAGTAATATCATA TTTTATTTCTCTTTCTTCTTTGTGTACTTATACTATGTTTCGTGTAATCAATCAACTCATAGTACACC (143) 3′SbfI MSP7 (SEQ ID NO: F88) GCGCATCCTGCAGGTTACATTGTGTTTAGTAAATTAAATGAATATTCTAAG (51) >gi|124800998|ref|XM_001349543.1| Plasmodium falciparum 3D7 merozoite surface protein 5 (MSP5) mRNA, complete cds (SEQ ID NO: F89) ATGAATATATTATGTATTCTATCATATATTTATTTCTTCGTTATTTTCTATAGTTTGAATTTAAATAATAAAAAT GAAAATTTTTTGGTTGTCAGAAGATTAATGAATGACGAAAAAGGAGAAGGTGGTTTTACAAGTAAAAATAAAGAG AATGGAAATAATAATAGAAATAATGAAAATGAACTAAAAGAAGAAGGTTCTTTACCTACTAAGATGAATGAAAAA AATTCCAATTCATCAGATAAACAGCCAAATGATATTTCACATGATGAATCAAAGAGCAATTCTAATAATTCACAA AATATCCAAAAAGAACCTGAAGAAAAAGAGAACAGTAACCCTAATTTAGATAGTAGTGAAAATTCGAGTGAAAGC GCAACACGTTCTGTTGATATATCAGAACATAATTCTAATAATCCAGAGACGAAAGAAGAGAATGGAGAAGAACCT TTAGATCTTGAAATTAATGAGAATGCAGAAATAGGTCAAGAACCTCCAAATAGATTACATTTTGACAATGTAGAT GATGAGGTGCCACATTATAGCGCCCTAAGATATAATAAAGTAGAAAAAAATGTAACCGATGAAATGTTATTATAT AATATGATGAGTGATCAAAATAGAAAATCATGTGCCATAAATAATGGTGGATGTTCTGATGATCAAATATGTATA AATATAAATAATATAGGAGTTAAATGTATATGTAAGGATGGATATTTACTTGGTACGAAATGTATAATATTGAAT TCTTATTCTTGCCATCCATTCTTCTCTATTCTTATTTATATTACATTGTTTTTGTTATTATTCGTTTAA 5′MfeI SYNPRO MSPS (SEQ ID NO: F90)

3′SbfI MSP5 (SEQ ID NO: F91) GCGCATCCTGCAGGTTAAACGAATAATAACAAAAACAATGTAATATAAATAAGAATAGAGAAGAATGGATGGC MSP3 (SEQ ID NO: F92) ATGAAAAGTTTTATAAATATTACTCTTTCATTATTCTTGTTACATTTATATATTTATATAAATAATGT TGCTAGTAAAGAAATTGTAAAAAAATATAATCTTAACTTAAGAAATGCAATATTGAATAATAATTCTC AAATAGAAAATGAAGAAAATGTAAATACTACAATTACTGGTAATGATTTTAGTGGTGGAGAATTCTTG TGGCCTGGTTATACGGAAGAATTAAAAGCTAAAAAAGCTTCCGAAGATGCTGAAAAAGCTGCTAATGA TGCTGAAAATGCTTCAAAAGAGGCAGAAGAAGCTGCTAAAGAAGCAGTAAATTTAAAGGAATCTGATA AATCTTATACAAAAGCAAAAGAAGCATGTACAGCTGCTTCAAAGGCAAAGAAAGCTGTTGAAACTGCT TTAAAGGCAAAAGATGATGCTGAAAAATCTTCAAAAGCTGATAGTATTTCTACAAAAACAAAAGAATA TGCTGAAAAAGCAAAAAATGCTTATGAAAAGGCAAAAAATGCTTATCAAAAAGCAAACCAAGCTGTTT TAAAAGCAAAAGAAGCTTCTAGTTATGATTATATTTTAGGTTGGGAATTTGGAGGAGGCGTTCCAGAA CACAAAAAAGAAGAAAATATGTTATCACATTTATATGTTTCTTCAAAGGATAAGGAAAATATATCTAA GGAAAATGATGATGTATTAGATGAGAAGGAAGAAGAGGCAGAAGAAACAGAAGAAGAAGAACTTGAAG AAAAAAATGAAGAAGAAACAGAATCAGAAATAAGTGAAGATGAAGAAGAAGAAGAAGAAGAAGAAGAA AAGGAAGAAGAAAATGACAAAAAAAAAGAACAAGAAAAAGAACAAAGTAATGAAAATAATGATCAAAA AAAAGATATGGAAGCACAGAATTTAATTTCTAAAAACCAGAATAATAATGAGAAAAACGTAAAAGAAG CTGCTGAAAGCATCATGAAAACTTTAGCTGGTTTAATCAAGGGAAATAATCAAATAGATTCTACCTTA AAAGATTTAGTAGAAGAATTATCCAAATATTTCAAAAATCATTAA 5′BamsynproMSP3 (SEQ ID NO: F93) atgCGCGGATCCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAAAATGAAAAGTTTTA TAAATATTACTCTTTCATTATTCTTGTTACATTTATATATTTATATAAATAATGTTGC 3′PstMSP3 (SEQ ID NO: F94) GCGCATCTGCAGTTAATGATTTTTGAAATATTTGGATAATTCTTCTAC 5′MSP3WPP (SEQ ID NO: F95) GGTAATGATTTTAGTGGTGGAGAATTCTTGTGGCCTGGTTATACGGAAGAATTAAAAGC 3′MSP3WPP (SEQ ID NO: F96) GCTTTTAATTCTTCCGTATAACCAGGCCACAAGAATTCTCCACCACTAAAATCATTACC Plasmodium falciparum 3D7 conserved Plasmodium protein, PfSEA-1, nucleotides 2431-3249 (PF10_0212a) mRNA, complete cds-825bp (SEQ ID NO: G1) ATGAACGAGGATAGAGGAATATACGATGAATTATTAGAAAATGATATGTGTGATTTATACAATTTAAAAATGCAT GATTTGCATAATTTAAAATCCTATGATTTTGGATTATCTAAAGATTTATTAAAAAAGGATATTTTCATATATAGT AATAATTTGAAAAATGATGATATGGATGATGATGATAATAATAATATGAATGATATTGCTATAGGTGAAAATGTA ATATATGAAAATGATATACATGAAAATAATATAGATGATAATGATATGTATAATAATTACGTGAATGGAAATGAT TTATATATTAACAATATGCAGGATGATGCCATGGACGATATTGTATATGATGAGGAAGAAATTAAAAGCTTCCTA GATAAATTAAAATCTGATATATCAAATCAAATGAATGTAAAAAATGGAAATGTCGAAGTTACAGGAAATGGTGGT AATGAAGAAATGTCTTATATAAATAATGATGAAAATTTACAAGCTTTTGATTTGTTAGATAATTTCCATATGGAT GATTATGGTAATAATTATAATGATAATGAAGAAGATGGGGATGGGGATGGGGATGACGATGAACAGAAGAAAAGA AAACAAAAAGAGTTACATAATGTAAATGGAAAATTAAACTTATCAGATTTAAATGAATTAAATGTAGATGATATA AATAATAATTTCTATATGTCAACTCCTCGAAAATCTATAGATGAACGTAAAGATACGGAATGTCAAACAGATTTT CCATTATTAGATGTATCAAGGAATACTAATAGGACTCCTAGAAGAAAAAGTGTGGAAGTAATACTTGTAGAATAA LSA-RPTLS of P. falciparum (SEQ ID NO: H1) GGTAAGATCATCAAGAACAGCGAGAAGGACGAAATCATCAAGAGCAACCTGCGTAGCGGTAGCAGCAA TAGCCGTAATCGCATCAACGAGGAAAAGCACGAGAAGAAACACGTGCTGAGCCACAACAGCTACGAGA AAACCAAGAACAACGAAAACAACAAGTTCTTTGACAAGGATAAGGAACTGACCATGAGCAACGTGAAG AACGTTAGCCAGACCAACTTCAAAAGCCTGCTGCGTAACCTGGGTGTTAGCGAGAACATCTTTCTGAA GGAAAACAAACTGAACAAGGAAGGCAAACTGATTGAGCACATCATTAACGACGATGACGATAAGAAAA AGTATATCAAGGGTCAGGACGAAAACCGTCAAGAGGATCTGGAACAGGAGCGTCTGGAGCAGCAAAGC GACCTGGAACAAGAGCGTCTGGCGAAAGAAAAGCTGCAGGAACGCCTGGCGAAGGAGAAGCTGCAAGA ACAACAAAGCGACCTGGAGGAGCGTAAAGCGGATACCAAAAAGAACCTGGAACGTAAAAAGGAGCACG GTGACGTGCTGGCGGAAGATCTGTACGGCCGTCTGGAAATCCCGGCGATTGAGCTGCCGAGCGAAAAC GAGCGTGGTTACTATATTCCGCACCAAAGCAGCCTGCCGCAGGACAACCGTGGCAACAGCCGTGATAG CAAGGAAATCAGCATCATTGAGAACACCAACCGTGAAAGCATCACCACCAACGTTGAGGGTCGTCGTG ACATTCACAAGGGCCACCTGGAGGAGAAGAAGGACGGTAGCATCAAACCGGAACAAAAAGAGGACAAA AGCGCGGATATCCAGAACCACACCCTGGAAACCGTGAACATTAGCGACGTTAACGATTTCCAAATCAG CAAGTACGAAGACGAGATTAGCGCGGAATATGACGATAGCCTGATTGATGAGGAAGAGGACGATGAAG ACCTGGATGAGTTCAAACCGATCGTGCAATACGACAACTTTCAGGATGAAGAGAACATCGGCATTTAT AAGGAACTGGAGGACCTGATTGAGAAGAAC GAGAACCTGGACGATCTGGATGAAGGTATCGAGAAGAGCAGCGAAGAGCTGAGCGAAGAGAAGATTAA AAAGGGCAAAAAGTACGAGAAGACCAAGGACAACAACTTCAAGCCGAACGACAAAAGCCTGTACGATG AACACATCAAAAAGTATAAGAACGACAAGCAGGTTAACAAGGAAAAGGAGAAGTTCATCAAAAGCCTG TTCCACATCTTCGACGGCGATAACGAAATTCTGCAAATCGTTGATGAACTGAGCGAAGACATTACCAA ATACTTTATGAAACTG 

What is claimed is:
 1. A method of eliciting a protective immune response in a subject against malaria infection, comprising administering to the subject an effective amount of a recombinant NYVAC (rNYVAC) virus capable of expressing a malaria antigen gene, wherein the malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: 1 and is inserted into a region of the NYVAC viral genome selected from the group consisting of A26L, A56R, I4L, J2R, B13/B14R, and C7L-K1L, and wherein the malaria antigen gene encodes a malaria antigen selected from the group consisting of a pre-erythrocytic stage antigen, a blood stage antigen, or a transmission blocking stage antigen.
 2. The method of claim 1, wherein administering the rNYVAC virus elicits a protective immune response against a poxvirus infection in the subject.
 3. The method of claim 1, wherein the rNYVAC virus is capable of expressing two or more malaria antigen genes encoding malaria antigens of different developmental stages.
 4. The method of claim 1, wherein two or more recombinant NYVAC viruses are administrated, and wherein the two or more rNYVAC viruses are capable of expressing two or more malaria antigen genes encoding malaria antigens of different developmental stages.
 5. The method of claim 4, wherein the rNYVAC virus is administered, either in combination or separately, via a route selected from the group consisting of scarification, intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, oral administration, and intranasal administration.
 6. The method of claim 5, wherein the rNYVAC virus is administered via scarification.
 7. The method of claim 1, wherein the rNYVAC virus is administered the subject as a vaccine formulation.
 8. The method of claim 1, wherein the malaria antigen gene is selected from the group consisting of AMA1, CelTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45 of Plasmodium falciparum, wherein the P. falciparum antigen gene is inserted into a region of the NYVAC viral genome selected from the group consisting of A26L, A56R, I4L, J2R, and B13/B14R regions, and wherein administering the recombinant NYVAC to a subject elicits a protective immune response against a P. falciparum infection in said subject.
 9. The method of claim 8, comprising administering to the subject an effective amount of five to thirty rNYVAC viruses, wherein the five to thirty rNYVAC viruses jointly express P. falciparum antigen genes of AMA1, CelTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45.
 10. The method of claim 9, wherein altogether 25 rNYVAC viruses are administered, wherein each of the 25 rNYVAC viruses express at least one different P. falciparum malaria antigen gene.
 11. The method of claim 8, comprising administering to the subject an effective amount of two to seven rNYVAC viruses, wherein the two to seven rNYVAC viruses jointly express at least P. falciparum antigens AMA1, CelTOS, LSA1-RPTLS, TRAP, and Pfs25.
 12. The method of claim 1, wherein the malaria antigen gene is selected from the group consisting of: CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25 of Plasmodium vivax, and wherein administering the rNYVAC viruses elicits a protective immune response against a P. vivax infection in the subject.
 13. The method of claim 12, comprising administering to the subject an effective amount of one to eight rNYVAC viruses, wherein the one to eight rNYVAC viruses jointly express P. vivax antigens of CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS
 25. 14. The method of claim 13, wherein altogether eight rNYVAC viruses are administered, wherein each of the one to eight recombinant NYVAC viruses individually expresses at least one different P. vivax malaria antigen.
 15. The method of claim 1, wherein the recombinant NYVAC virus is constructed by the steps of: a) preparing a shuttle plasmid comprising an expression cassette flanked by about 500 bp sequences upstream and downstream of the NYVAC A26L, A56R, I4L, J2R, B13/B14R, or C7L-K1L gene, wherein the expression cassette comprises the malaria antigen gene under the control of the compact synthetic early-late promoter; b) transfecting the shuttle plasmid to host mammalian cells and co-infecting the host mammalian cells with a parent NYVAC virus to allow in vivo recombination; and c) screening for a rNYVAC virus obtained from step b) to obtain the recombinant NYVAC virus, wherein the expression cassette is located in the rNYVAC genome.
 16. The method of claim 15, wherein the shuttle plasmid comprises an E. coli gpt gene.
 17. The method of claim 15, further comprising using a recombinant NYVAC virus as the parent NYVAC virus to obtain a second recombinant NYVAC virus capable of expressing two or more malaria antigen genes.
 18. A recombinant NYVAC virus capable of expressing a Plasmodium falciparum malaria antigen gene selected from the group consisting of: AMA1, CelTOS, CS, LSA1-RPTLS, SIAP1, SIAP2, SPATR, SPECT1, SPECT2, STARP, TRAP, EBA175 RII, MSP1-p19, MSP1-p42, MSP1DBL, MSP3, MSP5, MSP7, MSP9, PfSEA1, Rh5, Pfs16, Pfs25, Pfs28, and Pfs48.45, wherein the P. falciparum malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: 1 and is inserted into a region of the NYVAC viral genome selected from the group consisting of: A26L, A56R, I4L, J2R, and B13/B14R.
 19. The recombinant NYVAC virus of claim 18, which is capable of expressing up to five P. falciparum malaria antigen genes, each of which is inserted into a different region of the NYVAC viral genome.
 20. A vaccine formulation comprising the recombinant NYVAC virus of claim 18, wherein the vaccine formulation is capable of eliciting a protective immune response against a P. falciparum infection when administered to a subject.
 21. The vaccine formulation of claim 20, comprising twenty-five recombinant NYVAC viruses, each of which expresses at least one different P. falciparum malaria antigen gene.
 22. The vaccine formulation of claim 20, comprising two to seven recombinant NYVAC viruses, wherein the two to seven recombinant NYVAC viruses jointly express at least P. falciparum antigens AMA1, CelTOS, LSA1-RPTLS, TRAP, and Pfs25.
 23. A recombinant NYVAC virus capable of expressing a Plasmodium vivax malaria antigen gene selected from the group consisting of: CS-VK210, CS-VK247, AMA1, TRAP-SSP2, MSP1 fragment p42, Duffy Binding Protein region II, PVS 28, and PVS 25, wherein the P. vivax malaria antigen gene is under the control of a compact synthetic early-late promoter comprising the nucleotide sequence of SEQ ID NO: 1 and is inserted into a region of the NYVAC viral genome selected from the group consisting of A26L, A56R, I4L, J2R, B13/B14R, and C7L-K1L.
 24. The recombinant NYVAC virus of claim 23, capable of expressing eight P. vivax malaria antigen genes, wherein three P. vivax malaria antigen genes are inserted in C7L-K1L region of the NYVAC viral genome.
 25. A vaccine formulation comprising the recombinant NYVAC virus of claim 23, wherein the vaccine formulation is capable of eliciting a protective immune response against a P. vivax infection when administered to the subject.
 26. The vaccine formulation of claim 25, comprising eight recombinant NYVAC viruses, each of which expresses at least one different P. vivax malaria antigen gene. 